Catalytic de-crosslinking of samples for in situ analysis

ABSTRACT

The present disclosure relates in some aspects to methods and compositions for in situ analysis involving catalytic de-crosslinking of biological samples.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 63/353,506, filed Jun. 17, 2022, entitled “CATALYTIC DE-CROSSLINKINGOF SAMPLES FOR IN SITU ANALYSIS,” which is herein incorporated byreference in its entirety for all purposes.

FIELD

The present disclosure relates in some aspects to compositions andmethods for catalytic de-crosslinking of a fixed biological sample andfor preparing the biological sample for in situ analysis.

BACKGROUND

Methods are available for analyzing analytes such as nucleic acids andproteins present in a biological sample, e.g., a cell or tissue sample.Current methods for analyzing analytes in situ can have low sensitivityand specificity, have high background and/or low signal-to-noise ratio(e.g., due to autofluorescence), have limited plexity, or be biased,time-consuming, labor-intensive, and/or error-prone. Improved methodsfor analyzing analytes in a biological sample are needed. Providedherein are methods and compositions that meet such and other needs.

SUMMARY

Nucleic acid probe-based assay methods for in situ analysis such assingle molecule fluorescent hybridization (smFISH) have enablednanoscale-resolution imaging of RNA in cells and tissues. However, mostmethods of analyte detection are not compatible with fixed tissueswithout specific sample preparation to clear crosslinking and renderanalytes accessible to biochemical reactions. In one aspect, backgroundautofluorescence can arise from and/or be exacerbated by samplefixation/crosslinking, such as formalin-fixation. Backgroundautofluorescence may have a significant, ongoing impact on the abilityto detect and resolve fluorescence signals from analytes of interestover other components also present in the biological samples, especiallywhen analysis is carried out over multiple rounds of imaging. In anotheraspect, molecular crosslinks may render analytes (e.g., nucleic acidsequences, epitopes, and/or antigens) less accessible to detectionreagents such as labelling agents (e.g., nucleic acid probes targetingcellular DNA or RNA, or antibodies targeting protein analytes), therebyreducing detection efficiency and sensitivity. As such, de-crosslinkingfixed samples for in situ analysis is critical and improved methods areneeded.

In some embodiments, provided herein is a method comprising: a)providing a biological sample immobilized on a substrate, wherein thebiological sample is fixed; b) contacting the biological sample with acatalyst that catalyzes de-crosslinking of molecular crosslinks in thebiological sample; c) contacting the biological sample with a labellingagent that directly or indirectly binds to an analyte at a location inthe biological sample; and d) detecting an optical signal associatedwith the labelling agent or a product thereof, thereby detecting theanalyte at the location in the biological sample.

In some embodiments, disclosed herein is a method for sample analysis,comprising: a) providing a biological sample immobilized on a substrate,wherein the biological sample is fixed; b) contacting the biologicalsample with a catalyst that catalyzes de-crosslinking of molecularcrosslinks in the biological sample; c) contacting the biological samplewith a labelling agent that directly or indirectly binds to an analyteat a location in the biological sample; and d) detecting an opticalsignal associated with the labelling agent or a product thereof, therebydetecting the analyte at the location in the biological sample.

In some embodiments, the molecular crosslinks are products of one ormore crosslinking agents. In some embodiments, the one or morecrosslinking agents comprise an aldehyde, optionally wherein thecrosslinking agent comprises formaldehyde. In any of the embodimentsherein, the molecular crosslinks can be on RNA, DNA, protein,carbohydrate, lipid, and/or other molecules in the biological sample.

In any of the embodiments herein, the catalyst can catalyzede-crosslinking of inter-molecular crosslinks and/or intra-molecularcrosslinks in the biological sample, optionally wherein theinter-molecular crosslinks and/or intra-molecular crosslinks comprise anaminal bridge. In any of the embodiments herein, the catalyst can be awater-soluble catalyst. In any of the embodiments herein, the catalystcan be an organic molecule. In any of the embodiments herein, thecatalyst can be a transimination catalyst. In any of the embodimentsherein, the catalyst can catalyze de-crosslinking of aminal crosslinksin the biological sample. In any of the embodiments herein, the catalystcan catalyze breakdown of hemi-aminal adducts and/or aminal adducts inthe biological sample.

In any of the embodiments herein, the catalyst can be a compound offormula (I),

or a salt, zwitterion, or solvate thereof, wherein: A is selected fromthe group consisting of —COOH, —P(═O)(OH)₂, and S(═O)₂OH; X¹, X², X³,and X⁴ are each independently selected from the group consisting of: CH,CR^(a), and N; each occurrence of R^(a) is independently selected fromthe group consisting of C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, —NO₂,—NR′R″, and —C(═O)NR′R″; and each occurrence of R′ and R″ isindependently selected from the group consisting of H and C₁₋₆ alkylwhich is optionally substituted with

wherein n1 is an integer from 12 to 16.

In any of the preceding embodiments, the catalyst can comprise one ormore compounds selected from the group consisting of

or a salt, zwitterion, or solvate thereof. In any of the precedingembodiments, the catalyst can comprise

or a salt, zwitterion, or solvate thereof. In any of the precedingembodiments, the catalyst can comprise

or a salt, zwitterion, or solvate thereof. In any of the precedingembodiments, the catalyst can comprise

or a salt, zwitterion, or solvate thereof. In any of the precedingembodiments, the catalyst can comprise

or a combination thereof, or a salt, zwitterion, or solvate thereof. Inany of the preceding embodiments, the catalyst can comprise

or a salt, zwitterion, or solvate thereof. In any of the precedingembodiments, the catalyst can comprise

or a salt, zwitterion, or solvate thereof.

In any of the embodiments herein, the catalyst can be a compound offormula (II),

or a salt, zwitterion, or solvate thereof, wherein: L¹ is selected fromthe group consisting of —O—, —N(H)—, —N(C₁₋₃ alkyl)-,—N(CH₂CH₂O)₁₋₁₀—CH₃—, —S(O)₀₋₂—, —CH₂—, and a bond; R¹ is selected fromthe group consisting of: H; C₁₋₆ alkyl; C₁₋₆ haloalkyl; C₆₋₁₀ aryloptionally substituted with 1-4 R^(b); and 5- to 10-membered heteroaryl,wherein 1-4 ring atoms are heteroatoms each independently selected fromthe group consisting of: N, N(H), N(C₁₋₃ alkyl), O, and S, wherein theheteroaryl is optionally substituted with 1-4 independently selectedR^(b); and each R^(b) is independently selected from the groupconsisting of: halo, cyano, —OH, —NH₂, —NH(C₁₋₃ alkyl), —N(C₁₋₃ alkyl)₂,C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₄ alkoxy, and C₁₋₄ haloalkoxy.

In some embodiments, the catalyst comprises

or a salt, zwitterion, or solvate thereof.

In any of the embodiments herein, the substrate can comprise a planarsurface configured to contact the biological sample. In any of theembodiments herein, the substrate may but does not need to comprise abead, particle, or microwell. In any of the preceding embodiments, thesubstrate can be transparent. In any of the preceding embodiments, thesubstrate can be a glass slide or a plastic slide. In any of thepreceding embodiments, the substrate may but does not need to comprisenucleic acid immobilized thereon prior to contacting the biologicalsample.

In any of the preceding embodiments, the biological sample can be atissue section. In any of the preceding embodiments, the biologicalsample can comprise cells immobilized on the substrate. In someembodiments, the cells are dissociated cells, cultured cells, and/orcells isolated from a subject. In any of the preceding embodiments, thebiological sample can be an aldehyde-fixed biological sample. In any ofthe preceding embodiments, the biological sample can be aformaldehyde-fixed biological sample. In any of the precedingembodiments, the biological sample can be a paraffinzed biologicalsample. In some embodiments, the biological sample is aformaldehyde-fixed paraffin-embedded (FFPE) biological sample. In any ofthe preceding embodiments, the biological sample can be a fresh frozenbiological sample that has been crosslinked.

In any of the preceding embodiments, the method can comprise, prior tocontacting the biological sample with the catalyst, a step ofdehydrating the biological sample. In some embodiments, the dehydratingstep comprises drying the biological sample at 42° C. for 3 hours ordrying the biological sample at room temperature overnight. In any ofthe preceding embodiments, the method can comprise, prior to contactingthe biological sample with the catalyst, a step of baking the biologicalsample. In some embodiments, the baking step comprises baking thebiological sample uncovered at 60° C. for 2 hours. In any of thepreceding embodiments, the method can comprise, prior to contacting thebiological sample with the catalyst, a step of de-paraffinizing thebiological sample. In some embodiments, the de-paraffinizing comprisescontacting the biological sample with xylene, ethanol, and water, or,sequentially contacting the biological sample with xylene, absoluteethanol, about 96% ethanol, and about 70% ethanol. In any of thepreceding embodiments, the method can comprise, prior to contacting thebiological sample with the catalyst, a step of re-hydrating thebiological sample. In some embodiments, the re-hydrating comprisessequentially contacting the biological sample with 100% ethanol, 100%ethanol, 96% ethanol, 70% ethanol, each for 3 minutes, followed bycontacting the biological sample with nuclease free water for 20seconds.

In any of the preceding embodiments, the method can comprise, prior tocontacting the biological sample with the catalyst, a step ofpretreating the biological sample. In some embodiments, the pretreatingcomprises contacting the biological sample with a proteinase. In someembodiments, the proteinase is a collagenase. In some embodiments, theproteinase is present in a solution or suspension comprising a buffer.In any of the preceding embodiments, the method can comprise, prior tocontacting the biological sample with the catalyst, a step ofpermeabilizing the biological sample. In any of the precedingembodiments, the method can comprise, prior to contacting the biologicalsample with the catalyst, a step of staining the biological sample andimaging the stained biological sample. In some embodiments the stainingcomprises the use of a histological stain and/or an immunological stain.

In any of the preceding embodiments, the catalyst can be contacted withthe biological sample at a concentration between about 5 mM and about500 mM. In any of the preceding embodiments, the catalyst can becontacted with the biological sample at a concentration between about 10mM and about 400 mM. In any of the preceding embodiments, the catalystcan be contacted with the biological sample at a concentration betweenabout 50 mM and about 300 mM. In any of the preceding embodiments, thecatalyst can be contacted with the biological sample at a concentrationbetween about 75 mM and about 250 mM In any of the precedingembodiments, the catalyst can be contacted with the biological sample ata concentration between about 100 mM and about 200 mM.

In any of the preceding embodiments, the catalyst can be contacted withthe biological sample for about 1 minute to about 150 minutes. In any ofthe preceding embodiments, the catalyst can be contacted with thebiological sample for about 5 minute to about 100 minutes. In any of thepreceding embodiments, the catalyst can be contacted with the biologicalsample for about 10 minute to about 50 minutes. In any of the precedingembodiments, the catalyst can be contacted with the biological samplefor about 15 minute to about 30 minutes.

In any of the preceding embodiments, the catalyst can be contacted withthe biological sample at a temperature between about 5° C. and about100° C. In any of the preceding embodiments, the catalyst can becontacted with the biological sample at a temperature between about 50°C. and about 95° C. In any of the preceding embodiments, the catalystcan be contacted with the biological sample at a temperature betweenabout 75° C. and about 90° C. In some embodiments, the catalyst iscontacted with the biological sample at 80° C. for 30 minutes.

In any of the preceding embodiments, a solution or a suspensioncomprising the catalyst and a buffer can contacted with the biologicalsample. In some embodiments, the buffer comprises citrate,tris(hydroxymethyl)aminomethane (Tris), phosphate-buffered saline (PBS),2-[4-˜(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES),2-(N-morpholino)ethanesulfonic acid (IMES), or a combination thereof. Inany of the preceding embodiments, the buffer can comprise dimethylsulfoxide (DMSO). In any of the preceding embodiments, the buffer cancomprise Tris and a chelating agent, optionally wherein the chelatingagent is ethylenediaminetetraacetic acid (EDTA) and the buffer isTris-EDTA (TE).

In any of the preceding embodiments, the buffer can be present in thesolution or suspension at a concentration between about 5 mM and about250 mM. In any of the preceding embodiments, the buffer can be presentin the solution or suspension at a concentration between about 100 mMand about 200 mM In any of the preceding embodiments, the solution orsuspension can have a pH between about 4 and about 10, In any of thepreceding embodiments, the solution or suspension can have a pH betweenabout 6 and about 8.

In any of the preceding embodiments, the solution or suspension cancomprise sodium dodecyl sulfate (SDS), urea, and/or a proteinase. Insome embodiments, the proteinase is proteinase K. In any of thepreceding embodiments, the solution or suspension can comprise sodiumdodecyl sulfate (SDS) and proteinase K. In any of the precedingembodiments, the solution or suspension can comprise urea and proteinaseK. In some embodiments, the urea concentration is between about 0.01 Mand about 1 M. In some embodiments, the proteinase K concentration isbetween about 0.1 μg/mL and about 2 μg/mL. In some embodiments, the ureaconcentration is about 0.5 M and the proteinase K concentration isbetween about 0.5 μg/mL and about 1 μg/mL.

In any of the preceding embodiments, contacting the biological samplewith the catalyst can substantially preserve adhesion of the biologicalsample to the substrate, and/or contacting the biological sample withthe catalyst can substantially preserve integrity of the biologicalsample.

In any of the preceding embodiments, the method can comprise: aftercontacting the biological sample with the catalyst, a step of washingthe biological sample, optionally wherein the washing comprises washingthe biological sample in phosphate-buffered saline with Tween detergent(PBST) for 1 minute for three times: a step of permeabilizing thebiological sample before, during, and/or after contacting the biologicalsample with the catalyst; and/or a step of staining the biologicalsample and imaging the stained biological sample, optionally wherein thestaining comprises the use of a histological stain and/or animmunological stain.

In any of the preceding embodiments, the analyte or product thereof canremain in the biological sample during the contacting with the catalyst,during the contacting with the labelling agent, and during detecting theoptical signal; and/or the analyte or product thereof. Can substantiallyremain at the location during the contacting with the catalyst, duringthe contacting with the labelling agent, and during detecting theoptical signal.

In any of the preceding embodiments, the method nay but does not need tocomprise migrating the analyte or a product thereof towards thesubstrate. In some embodiments, the migration is passive migration oractive migration. In any of the preceding embodiments, the method maybut does not need to comprise migrating the analyte or a product thereofoutside the biological sample. In any of the preceding embodiments, themethod may but does not need to comprise capturing the analyte or aproduct thereof by a capture agent immobilized on the substrate.

In any of the preceding embodiments, the labelling agent may but doesnot need to be immobilized on the substrate prior to contacting thebiological sample. In any of the preceding embodiments, a solution or asuspension comprising the labelling agent can be contacted with thebiological sample. In any of the preceding embodiments, the labellingagent can comprise a binding moiety. In some embodiments, the bindingmoiety comprises a nucleic acid or an antibody or epitope bindingfragment thereof. In any of the preceding embodiments, the labellingagent can comprise a detectable label. In some embodiments, thedetectable label comprises a nucleic acid or an optically detectablelabel. In any of the preceding embodiments, the labelling agent cancomprise a reporter oligonucleotide. In some embodiments, the reporteroligonucleotide comprises a barcode sequence.

In any of the preceding embodiments, the analyte is a cellular nucleicacid. In some embodiments, the cellular nucleic acid is genomic DNA,mRNA, or cDNA. In some embodiments, the labelling agent is a primaryprobe that hybridizes to the cellular nucleic acid. In some embodiments,the primary probe comprises a barcode sequence. In any of the precedingembodiments, the primary probe can be selected from the group consistingof: a primary probe comprising a 3′ or 5′ overhang upon hybridization tothe cellular nucleic acid, optionally wherein the 3′ or 5′ overhangcomprises one or more barcode sequences; a primary probe comprising a 3′overhang and a 5′ overhang upon hybridization to the cellular nucleicacid, optionally wherein the 3′ overhang and the 5′ overhang eachindependently comprises one or more barcode sequences; a circularprimary probe; a circularizable primary probe or probe set; a primaryprobe or probe set comprising a split hybridization region configured tohybridize to a splint, optionally wherein the split hybridization regioncomprises one or more barcode sequences; and a combination thereof. Insome embodiments, the labelling agent is a detectable probe thathybridizes to a primary probe or a product or complex thereof, whereinthe primary probe hybridizes to the cellular nucleic acid. In someembodiments, the product or complex of the primary probe is selectedfrom the group consisting of: a rolling circle amplification (RCA)product, a complex comprising an initiator and an amplifier forhybridization chain reaction (HCR), a complex comprising an initiatorand an amplifier for linear oligonucleotide hybridization chain reaction(LO-HCR), a primer exchange reaction (PER) product, and a complexcomprising a pre-amplifier and an amplifier for branched DNA (bDNA). Inany of the preceding embodiments, the detectable probe can hybridize toa barcode sequence in the primary probe or product or complex thereof.In any of the preceding embodiments, the detectable probe can comprise abarcode sequence in a region that does not hybridize to the primaryprobe or product or complex thereof. In any of the precedingembodiments, the detectable probe can be selected from the groupconsisting of: a detectable probe comprising a 3′ or 5′ overhang uponhybridization to the primary probe or product or complex thereof,optionally wherein the 3′ or 5′ overhang comprises one or more barcodesequences; a detectable probe comprising a 3′ overhang and a 5′ overhangupon hybridization to the primary probe or product or complex thereof,optionally wherein the 3′ overhang and the 5′ overhang eachindependently comprises one or more barcode sequences; a circulardetectable probe; a circularizable detectable probe or probe set; adetectable probe or probe set comprising a split hybridization regionconfigured to hybridize to a splint, optionally wherein the splithybridization region comprises one or more barcode sequences; and acombination thereof. In any of the preceding embodiments, the detectableprobe can comprise a fluorescent label. In any of the precedingembodiments, the detectable probe can comprise a region for binding to afluorescently labelled probe.

In any of the preceding embodiments, the analyte can comprise anon-nucleic acid moiety, optionally wherein the non-nucleic acid moietyis a protein, a carbohydrate, a lipid, a small molecule, or a complexthereof. In some embodiments, the labelling agent comprises i) ananalyte-binding region that directly or indirectly binds to thenon-nucleic acid moiety and ii) a reporter oligonucleotide, optionallywherein the analyte-binding region is an antibody or epitope bindingfragment thereof.

In any of the preceding embodiments, the method can comprise: contactingthe biological sample with a first labelling agent that directly orindirectly binds to a nucleic acid analyte at a first location in thebiological sample, and detecting a first optical signal associated withthe first labelling agent or a product thereof, and contacting thebiological sample with a second labelling agent that directly orindirectly binds to a protein analyte at a second location in thebiological sample, and detecting a second optical signal associated withthe second labelling agent or a product thereof, thereby detecting thenucleic acid analyte and the protein analyte at the first and secondlocations in the biological sample, respectively.

In some embodiments, the nucleic acid analyte is an mRNA, and theprotein analyte is an intracellular protein, a membrane-bound protein,or an extracellular protein. In any of the preceding embodiments, thefirst and second locations can be the same location or differentlocations.

In any of the preceding embodiments, the product of the labelling agentcan be generated in situ in the biological sample. In any of thepreceding embodiments, the optical signal can be detected in situ in thebiological sample. In any of the preceding embodiments, the opticalsignal can be detected by imaging the biological sample. In someembodiments, the imaging comprises fluorescent microscopy.

In any of the preceding embodiments, the number of optical signalsdetected in a unit area in the biological sample can be greater thanthat without contacting the biological sample with the catalyst. In anyof the preceding embodiments, the analyte comprises a nucleic acid andthe number of optical signals detected per unit nuclei area in thebiological sample can be greater than that without contacting thebiological sample with the catalyst. In any of the precedingembodiments, the intensity of the optical signal detected in thebiological sample can be greater than that without contacting thebiological sample with the catalyst. In any of the precedingembodiments, the signal-to-noise ratio of the optical signal detected inthe biological sample can be greater than that without contacting thebiological sample with the catalyst.

In some aspects, disclosed herein is a method for sample analysis,comprising: a) contacting a biological sample with a catalyst thatcatalyzes de-crosslinking of molecular crosslinks in the biologicalsample, wherein the biological sample is a formaldehyde-fixed biologicalsample immobilized on a substrate before contacting with the catalyst,and wherein the catalyst is a compound of formula (I),

or a salt, zwitterion, or solvate thereof, wherein: A is selected fromthe group consisting of —COOH, —P(═O)(OH)₂, and S(═O)₂OH; X¹, X², X³,and X⁴ are each independently selected from the group consisting of: CH,CR^(a), and N; each occurrence of R^(a) is independently selected fromthe group consisting of C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, —NO₂,—NR′R″, and —C(═O)NR′R″; and each occurrence of R′ and R″ isindependently selected from the group consisting of H and C₁₋₆ alkylwhich is optionally substituted with

wherein n1 is an integer from 12 to 16; b) contacting the biologicalsample with a detectable probe that directly or indirectly binds to anucleic acid at a first location in the biological sample; and c)detecting an optical signal associated with the detectable probe,thereby detecting the nucleic acid at the first location in thebiological sample.

In some embodiments, the biological sample is contacted with a solutionor suspension comprising the catalyst in a citrate buffer between pH 5and pH 7. In some embodiments, the biological sample is contacted with asolution or suspension comprising the catalyst in a TE buffer between pH8.5 and pH 9.5. In some embodiments, the biological sample is contactedwith a solution or suspension comprising the catalyst in a PBS bufferbetween pH 6.8 and pH 8.0. In any of the preceding embodiments, thecatalyst concentration in the solution or suspension can be betweenabout 50 mM and about 400 mM. In any of the preceding embodiments, thecatalyst concentration in the solution or suspension can be betweenabout 100 mM and about 200 mM.

In any of the preceding embodiments, the biological sample can becatalytically de-crosslinked at a temperature between about 60° C. andabout 95° C. In any of the preceding embodiments, the biological samplecan be catalytically de-crosslinked at a temperature between about 70°C. and about 90° C. In any of the preceding embodiments, the biologicalsample can be catalytically de-crosslinked at a temperature betweenabout 75° C. and about 85° C. In any of the preceding embodiments, thebiological sample can be catalytically de-crosslinked at a temperaturebetween about 75° C. and about 85° C. In any of the precedingembodiments, the biological sample can be catalytically de-crosslinkedfor about 5 minutes to about 1 hour. In any of the precedingembodiments, the biological sample can be catalytically de-crosslinkedfor about 10 minutes to about 45 minutes. In any of the precedingembodiments, the biological sample can be catalytically de-crosslinkedfor about 15 minutes to about 30 minutes.

In any of the preceding embodiments, the nucleic acid can be an RNA inthe biological sample. In any of the preceding embodiments, the nucleicacid can be a nucleic acid probe that directly or indirectly binds to anRNA in the biological sample. In any of the preceding embodiments, thenucleic acid can be a product of an RNA in the biological sample,optionally wherein the nucleic acid is a cDNA of the RNA. In any of thepreceding embodiments, the nucleic acid can be a product of a nucleicacid probe that directly or indirectly binds to an RNA in the biologicalsample. In some embodiments, the nucleic acid is a rolling circleamplification product (RCP).

In any of the preceding embodiments, the method can comprise: d)contacting the biological sample with a detectably labelled antibody oran epitope binding fragment thereof that binds to a polypeptide orcomplex thereof at a second location in the biological sample; and e)detecting an optical signal associated with the detectably labelledantibody, thereby detecting the polypeptide or complex thereof at thesecond location in the biological sample. In some embodiments, thedetectably labelled antibody comprises a fluorophore. In someembodiments, the detectably labelled antibody comprises a reporteroligonucleotide. In some embodiments, the optical signal associated withthe detectably labelled antibody is detected using a detectable probethat directly or indirectly binds to the reporter oligonucleotide.

Provided herein is a method for sample analysis which includescontacting a biological sample with a catalyst that catalyzesde-crosslinking of molecular crosslinks in the biological sample,wherein the biological sample is a sectioned formaldehyde-fixed cell ortissue sample immobilized on a substrate before contacting with thecatalyst; contacting the biological sample with a detectable probe thatdirectly or indirectly binds to an RNA analyte at a first location inthe biological sample; detecting an optical signal associated with thedetectable probe, thereby detecting the RNA analyte at the firstlocation in the biological sample; contacting the biological sample witha detectably labelled antibody or an epitope binding fragment thereofthat binds to a polypeptide or complex thereof at a second location inthe biological sample; and detecting an optical signal associated withthe detectably labelled antibody, thereby detecting the polypeptide orcomplex thereof at the second location in the biological sample. In someinstances, the optical signals are detected by imaging the biologicalsample on the substrate. In some embodiments, the catalyst comprises

or a salt, zwitterion, or solvate thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings illustrate certain features and advantages of thisdisclosure. These embodiments are not intended to limit the scope of theappended claims in any manner.

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 depicts the workflow of an exemplary method disclosed herein.

FIGS. 2A-2B depict exemplary reactions of fixation chemistry andreversal of molecular crosslinks. FIG. 2A shows formaldehyde fixationaffords stable, inert crosslinks between amine residues (e.g., aminalbond, CH₂-linked amine). FIG. 2B depicts an example of catalyticreversal of aminal crosslinks using a combination of acid catalysis andnucleophilic catalysis.

FIG. 3A depicts structures of an exemplary catalyst. In some aspects,with varying R groups, the pK_(a2) does not change significantly but thenucleophilicity (N) of —NH₂ does. FIG. 3B depicts structures of anexemplary catalyst under different pH and examples of pH-dependent acidcatalysis. In this example, both compound A and compound B exist insolution at pH 6.8, but only compound B with —COOH catalyzesde-crosslinking reaction. However, at pH 9, the compound exists mostlyas a deprotonated form (compound A), which does not catalyze thede-crosslinking reaction. FIG. 3C depicts exemplary phosphonic acidcatalysts which perform well at pH 6.8 since —OH of the phosphonic acidexists in solution to catalyze a de-crosslinking reaction.

FIG. 4 shows detected rolling circle amplification product (RCP) signaldensity (count/μm² nuclei area) in catalytically de-crosslinked humanbreast cancer samples compared to control samples de-crosslinked using asteamer.

FIGS. 5A-5B show representative images of anti-panCK antibody stainingin FFPE human breast cancer samples catalytically de-crosslinked incitrate buffer (FIG. 5A) or TE buffer (FIG. 5B), compared to a controlsample de-crosslinked using a steamer.

FIG. 6 shows representative DPAI images of the de-crosslinked FFPE humanbreast cancer samples.

FIG. 7A shows representative images of catalytically de-crosslinked FFPEhuman breast cancer, melanoma, lymph node, lung cancer, and normal lungtissue samples analyzed using detectable probes targeting RCPsassociated with GAPDH/RPLPO in the samples. FIG. 7B shows representativeimages of catalytically de-crosslinked FFPE human breast cancer,melanoma, lymph node, lung cancer, and normal lung tissue samplesstained with an anti-Vimentin antibody. FIG. 7C shows overlaid images ofnucleic acid detection and protein detection across the tissue types.

FIG. 8 shows representative images with detected RCP signals (leftpanel) and detected RCP signal intensity above local background (mean)(right panel) in catalytically de-crosslinked FFPE normal lung tissuesamples.

FIG. 9 shows representative images with detected RCP signals anddetected RCP signal intensity above local background (mean) incatalytically de-crosslinked FFPE mouse brain tissue sections.

FIG. 10A-10C shows detected rolling circle amplification product (RCP)signal density (count/μm² nuclei area) in catalytically de-crosslinkedhuman breast cancer samples with Compound 1 and various additives (e.g.,SDS, proteinase K, and/or urea) compared to control samplesde-crosslinked using Compound 1 only.

DETAILED DESCRIPTION

All publications, comprising patent documents, scientific articles anddatabases, referred to in this application are incorporated by referencein their entirety for all purposes to the same extent as if eachindividual publication were individually incorporated by reference. If adefinition set forth herein is contrary to or otherwise inconsistentwith a definition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth herein prevails over the definitionthat is incorporated herein by reference.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

I. Overview

Compositions and methods are needed for analyzing the spatiallocalization of analytes in a biological sample, such as variousarchived tissue materials comprising molecular crosslinks.Formalin/formaldehyde fixation is a dominant method for storage ofprimary tissue samples for pathology and life science research due toits robustness in maintaining tissue architecture at even ambienttemperatures. However, most methods of analyte detection are notcompatible with fixed tissues without specific sample preparation toclear crosslinking and render analytes accessible to biochemicalreactions, including those amenable to signal detection and/oramplification. Antigen retrieval is commonly employed using acombination of heat, temperature, buffer, and sometimes pressure toexpose antigens for more effective antibody staining, and proteinasedigestion or other forms of de-crosslinking are employed to enableeffective nucleic acid detection.

In some embodiments, provided herein are compositions and methods thatinvolve catalytic de-crosslinking for preparing samples, such as FFPEcell and tissue samples. In some embodiments, a sample such as anotherwise inaccessible fixed or FFPE sample can be treated with a buffercomprising a catalyst or a precursor thereof to provide accessibility oftarget analyte molecules in the sample for in situ analysis. In someembodiments, a sample such as an FFPE sample can be de-paraffinized. Insome embodiments, the de-paraffinized sample can be contacted with abuffer (e.g., a citrate buffer) comprising an effective concentration ofa catalyst or a precursor thereof for a period of time. In someembodiments, the sample can be incubated in a buffer together with thecatalyst, e.g., a heated buffer. In some embodiments, the buffer canhave antigen retrieving effect. In some embodiments, the buffer canfacilitate and/or promote catalytic de-crosslinking by the catalyst. Insome embodiments, antigen retrieving due to catalytic de-crosslinkingcan be combined with heating the sample and/or the buffer (e.g., in athermocycler), and/or be combined with an antigen retrieving effect ofthe buffer. In some embodiments, the sample can be washed using abuffer, such as a heated antigen retriever buffer or another buffer toremove products of the de-crosslinking reaction. After thede-crosslinking incubation and/or the de-crosslinking wash, the samplecan be subjected to probe/antibody binding and downstream imaging andanalysis.

In some embodiments, provided herein are various catalysts, buffercompositions comprising one or more catalysts, combinations of one ormore catalysts with one or more other de-crosslinking agents includingenzymes/proteases, and/or compositions comprising crowding agents (e.g.,PEG20k MW, to mitigate potential RNA loss during de-crosslinking). Insome embodiments, provided herein are methods involving catalyticde-crosslinking using any one or more of the compositions disclosedherein. In some embodiments, provided herein are methods involvingcatalytic de-crosslinking for various time periods and/or at varioustemperatures, optionally with heating and/or one or morepost-de-crosslinking washes. In some embodiments, the temperature of thesample can be adjusted as needed prior to, during, and/or afterde-crosslinking, for example, by controlling the rate of thermocyclerheating up/down.

In some embodiments, the methods can optionally include staining and/orimaging of the fixed biological sample (e.g., tissue section), of thede-crosslinked biological sample (e.g., tissue section), or both. Astain can be any appropriate stain, such as a histological stain (e.g.,hematoxylin and eosin) or an immunological stain (e.g., animmunofluorescent stain), or any other stain described herein, e.g., inSection IV-(v). Staining (e.g., H&E staining) and/or imaging can beperformed before and/or after the de-crosslinked biological sample(e.g., tissue section) is contacted with one or more nucleic acid probesand/or labelling agents (e.g., fluorescently labelled antibodies) andsignals associated with the nucleic acid probes and/or labelling agentsare detected in the sample.

FIG. 1 shows an exemplary method where a fixed sample immobilized on asubstrate is provided in step 101, and the sample can be optionallystained and/or imaged in step 102. If the sample is paraffin-embedded, ade-paraffinization and re-hydration step can be performed in step 103 toprepare the sample for catalytic de-crosslinking. The sample can againbe optionally stained and/or imaged in step 104 prior to de-crosslinkingin a buffer comprising a catalyst disclosed herein in step 105. Thesample can be optionally stained and/or imaged after catalyticde-crosslinking in step 106. A signal associated with a first analyte(e.g., nucleic acid or protein) in the de-crosslinked sample can bedetected in step 107, for instance, signals associated with the firstanalyte can be detected in sequential cycles using detection reagents(e.g., nucleic acid probes). A signal associated with a first analyte(e.g., nucleic acid or protein) in the de-crosslinked sample can bedetected in step 108, and in some examples, the first analyte can be acellular nucleic acid (e.g., genomic DNA, mRNA, or cDNA) and the secondanalyte can be a protein. The sample can be optionally stained and/orimaged after analyte detection in step 109.

Catalytic de-crosslinking disclosed herein can improve detection ofsignals associated with nucleic acid analytes and/or non-nucleic acidanalytes (e.g., protein analytes) in a fixed biological sampleimmobilized on a substrate (for instance, as demonstrated in FIG. 4 ,FIGS. 5A-5B, FIGS. 7A-7C, FIG. 8 , and FIG. 9 ) while substantiallymaintaining or improving sample integrity and/or adhesion to thesubstrate (for instance, as shown in FIG. 6 ), as compared to samplede-crosslinking without using the catalyst (e.g., using heating in asteamer). As such, signals associated with the analytes at multiplelocations in the catalytically de-crosslinked sample can be detectedmore efficiently (e.g., more signals can be detected in a given sample)and more accurately (e.g., with higher signal-to-noise ratios). Forexample, detection of signals associated with analytes can be probed anddetected (as described in Section III) in a catalytically de-crosslinkedsample (e.g., as described in Section II).

II. Catalytic De-Crosslinking

In some embodiments, provided herein are methods and compositions forproviding a fixed biological sample immobilized on a substrate, and forcatalytically de-crosslinking molecular crosslinks in the fixedbiological sample. In some embodiments, a method disclosed hereincomprises contacting a fixed biological sample with a compositioncomprising a catalyst or a precursor thereof.

A. Fixed Biological Samples

A biological sample disclosed herein can include any sample comprising acell, a tissue, or a derivative of a cell or a tissue. In someembodiments, a biological sample herein includes a fixed cell or tissuesample comprising molecular crosslinks that can be catalyticallyde-crosslinked using a catalyst disclosed herein. The ability to use afixed biological sample in an analytical method, such as in situanalysis of biological molecules (e.g., genomic DNA, RNA, cDNA, and/orproteins), is enhanced if the cross-links established during fixation ofthe biological sample are reversed so that an assay can be carried outbefore sample degradation occurs. In some aspects, data obtained from ade-crosslinked biological sample are similar to that obtained from afresh sample (e.g., a sample that is not fixed and/or crosslinked).

A fixed biological sample can be any appropriate fixed biologicalsample. In some embodiments, a fixed biological sample can be a fixedtissue sample (e.g., a fixed tissue section). In some embodiments, asample herein is not and does not comprise a dissociated tissue/cellsuspension. Molecules (e.g., analytes, labelling agents, nucleic acidprobes, etc., or products generated in situ in the sample) may but donot need to be removed from a sample herein for analysis before, during,or after catalytic de-crosslinking of the sample. In some embodiments,molecules (e.g., analytes, labelling agents, nucleic acid probes, etc.)are not removed from a sample herein for analysis. In some embodiments,signals associated with the molecules (e.g., analytes, labelling agents,nucleic acid probes, etc., or products generated in situ in the sample)are detected at multiple locations in the catalytically de-crosslinkedsample, e.g., the signals can be detected in situ in a catalyticallyde-crosslinked tissue section.

In some embodiments, the biological sample is fixed and the fixationcomprises contacting the sample with one or more agents that react withone another and/or with molecules in the biological sample. In someembodiments, the reaction creates molecular crosslinks between moleculesof the one or more agents, between molecules in the biological sample,and/or between molecules of the one or more agents and molecules in thebiological sample. In some embodiments, the one or more agents arecrosslinking agents, and the molecular crosslinks are products of one ormore reactions between a crosslinking agent and a molecule in thebiological sample.

In some embodiments, a biological sample is fixed using one or morecrosslinking agents comprising an aldehyde. In some embodiments, analdehyde includes a compound containing one or more aldehyde (—CHO)groups, where the aldehyde groups are capable of reacting with an amine(e.g., a primary amine, a secondary amine, or a tertiary amine) or withan amide. Amines are derivatives of ammonia, wherein one or morehydrogen atoms in amines have been replaced by a substituent such as analkyl or aryl group. These may respectively be called alkylamines andarylamines, and amines in which both types of substituent are attachedto one nitrogen atom may be called alkylarylamines. Exemplary aminesinclude amino acids (including amino acid residues of a protein havingside chains that can react with an aldehyde), biogenic amines,trimethylamine, and aniline. In some embodiments, molecular crosslinksin a fixed sample are formed via condensation between an aldehyde and anamine, and in some aspects, the condensation does not require heatingand/or an acidic condition. Amides having the structure R—CO—NR′R″ inwhich a nitrogen atom is attached to a carbonyl group. In someembodiments, molecular crosslinks in a fixed sample are formed viacondensation between an aldehyde and an amide, e.g., under heatingand/or acidic conditions. Exemplary aldehydes can include formaldehyde,paraformaldehyde, glutaraldehyde, glyoxal, and the like.

In some embodiments, fixing a biological sample comprises treating thesample with a crosslinking agent. In some embodiments, the crosslinkingagent comprises formaldehyde. Paraformaldehyde (PFA) is a polymer offormaldehyde. While paraformaldehyde itself is not a fixing agent, itcan be heated and/or treated under basic conditions until it becomessolubilized and broken down to formaldehyde molecules.

In some embodiments, the molecular crosslinks are on RNA, DNA, protein,carbohydrate, lipid, and/or other molecules in the biological sample. Insome embodiments, the molecular crosslinks comprise one or more aminalcrosslinks such as aminal bridges. In some embodiments, a fixedbiological sample can comprise aminal crosslinks among nucleic acids(e.g., genomic DNA, RNA such as mRNA, and/or cDNA), proteins,carbohydrates, lipids, and/or other molecules in the biological sample.Aminal crosslinks can be made, for example, by fixing a sample withformaldehyde.

In some embodiments, the fixative or fixation agent is formaldehyde.Formaldehyde as fixative comprises paraformaldehyde (or “PFA”) andformalin, both of which relate to the formaldehyde composition (e.g.,formalin is a mixture of formaldehyde and methanol). Thus, aformaldehyde-fixed biological sample may be formalin-fixed or PFA-fixed.Any suitable protocols and methods for the use of formaldehyde as afixation reagent to prepare fixed biological samples can be used in themethods and compositions of the present disclosure. In some embodiments,a biological sample is a formalin-fixed, paraffin-embedded (FFPE) tissuesample (e.g., an FFPE tissue section).

In some embodiments, aldehyde fixation methods can be combined withother tissue preservation methods. For example, aldehyde fixation can becombined with fresh frozen preservation of tissues, e.g., fresh frozentissues can be fixed using an aldehyde. Aldehyde fixation can also becombined with alcohol fixation, or with any number of commerciallyavailable fixation/preservation techniques. For example, aldehydefixation can be combined with salt-rich buffer solutions such asRNAlater™, low-temperature preservation buffers such as HypoThermosol,alcohol-PEG fixation (e.g., Neo-Rix, STATFIX, PAGA, UMFIX), PAXGene,Allprotect/Xprotect, CellCover, RN Assist, and/or zinc buffers.

In some embodiments, preparing fixed (e.g., aldehyde-fixed) biologicalsamples for in situ analysis may comprise catalytic de-crosslinkingdisclosed herein in combination with additional sample processing stepsand/or conditions before, during, and/or after catalyticde-crosslinking. Exemplary sample processing steps and/or conditions mayinclude longer permeabilization periods, additional permeabilizationreagents, or higher permeabilization reagent concentrations, e.g.,compared to samples that are not fixed, in order to allow detectionreagents (e.g., nucleic acid probes and/or antibodies or epitope-bindingfragments thereof) to bind to analytes in the sample.

In some embodiments, provided herein are methods of de-crosslinkingaminal crosslinks in a fixed biological sample. In some embodiments,provided herein are methods of in situ analysis using such ade-crosslinked sample. The methods described herein are not limited toany particular fixation reagent that results in crosslinks (e.g., aminalcrosslinks) and are equally amenable with any fixation method thatresults in intra-tissue crosslinking events (e.g., aminal intra tissuecrosslinking events).

In an exemplary fixation method, as shown in FIG. 2A, condensation of anamino group on a first molecule (e.g., nucleic acid or protein) in asample with formaldehyde can afford a reactive imine, which can reactwith a proximal amine (e.g., a CH₂-linked amine on a second molecule ofthe same or different species as the first molecule) to form an aminalbridge, thereby fixing the sample. While fixing can help stabilize thesample, molecular crosslinks could lead to antigen masking and/orbackground autofluorescence in the sample. For example, PFA inducedcrosslinks are known to be responsible for increased autofluorescence inFFPE tissues. In some cases, molecular crosslinks may block or restrictbiochemical reactions such as nucleic acid hybridization or methods ofsignal amplification utilized for analyte detection. Conventionalmethods for antigen retrieval may not sufficiently retrieve the maskedantigens and may not remove or reduce the background autofluorescencedue to fixing. The catalytic de-crosslinking methods disclosed hereinaddress these and other issues with conventional methods. In someaspects, catalytic de-crosslinking provided herein may revert thesemolecular crosslinks into native amines and thereby reduce theautofluorescence in the sample for in situ assay workflows (e.g.,imaging and detecting signals). In some cases, conventional antigenretrieval methods break these crosslinks but do not revert them intonative amines and may not be effective as the catalytic de-crosslinkingdescribed herein.

B. Preparing Samples for Catalytic De-Crosslinking

A biological sample can be immobilized on a substrate before, during,and/or after contacting with the catalyst. In some embodiments, thebiological sample is immobilized on the substrate before contacting withthe catalyst. In some embodiments, the biological sample remainsimmobilized on the substrate during and after contacting with thecatalyst. In some embodiments, the biological sample is immobilized onthe substrate after contacting with the catalyst. In some embodiments,the biological sample is immobilized on the substrate during contactingwith the catalyst. In some embodiments, a biological sample can beprovided in a fixed state. In some embodiments, a fixed biologicalsample can undergo one or more preparation steps before it is pretreatedand/or de-crosslinked.

In some embodiments, the substrate comprises a planar surface configuredto contact the biological sample and does not comprises a bead,particle, or microwell, optionally wherein the substrate is a glassslide or a plastic slide. In some embodiments, the substrate istransparent. In some embodiments, the substrate is suitable for imagingusing fluorescent microscopy, for instance, for in situ analytedetection, e.g., in situ sequencing or in situ sequential hybridization.In some embodiments, the substrate does not comprises nucleic acidimmobilized thereon prior to contacting the biological sample. In someembodiments, the biological sample is a tissue section. In someembodiments, the biological sample comprise cells immobilized on thesubstrate. In some embodiments, the cells are dissociated cells,cultured cells, and/or cells isolated from a subject. In someembodiments, the biological sample is an aldehyde-fixed biologicalsample. In some embodiments, the biological sample is aformaldehyde-fixed biological sample. In some embodiments, thebiological sample is a paraffinized biological sample. In someembodiments, the biological sample is a formaldehyde-fixedparaffin-embedded (FFPE) biological sample. In some embodiments, thebiological sample is a fresh frozen biological sample that has beencrosslinked.

In some embodiments, prior to contacting the biological sample with thecatalyst, the method comprises a step of pre-warming the biologicalsample. In some embodiments, a fixed biological sample (e.g., an FFPEtissue section) can be pre-warmed to between about 20° C. and about 60°C., e.g., about 30° C. to about 50° C., about 35° C. to about 45° C., orabout 40° C. to about 43° C. In some embodiments, the fixed biologicalsample can be pre-warmed by incubation in a water bath. In someembodiments, the fixed biological sample is a block of embedded tissue(e.g., formalin fixed and paraffin embedded) that can be sliced using amicrotome to generate embedded tissue sections, e.g., about 5 μm inthickness. In some embodiments, the microtome can be pre-warmed tobetween about 40° C. and about 43° C. for slicing the fixed biologicalsample.

In some embodiments, prior to contacting the biological sample with thecatalyst, the method comprises a step of dehydrating the biologicalsample. In some such embodiments, the fixed biological is dehydrated bydrying at a temperature higher than room temperature, e.g., at about 20°C. to about 60° C., about 30° C. to about 50° C., about 35° C. to about45° C., or about 40° C. to about 43° C., such as at about 40° C., 41°C., 42° C., 43° C., 44° C., or 45° C., for a period of time (e.g., about30 minutes to about 6 hours, about 1 hour to about 5 hours, about 2hours to about 4 hours, or about 3 hours). In some such embodiments, thefixed biological is dried at room temperature for a period of time(e.g., about 2 hours to about 24 hours, about 5 hour to about 20 hours,about 8 hours to about 16 hours, or overnight), for example, in adesiccator. In some such embodiments, the fixed biological is dried at atemperature higher than room temperature, followed by drying at roomtemperature.

In some embodiments, the method comprises, prior to contacting thebiological sample with the catalyst, a step of baking the biologicalsample. In some such embodiments, the fixed biological is baked at atemperature, e.g., at about 40° C. to about 80° C., about 45° C. toabout 75° C., about 50° C. to about 70° C., or about 55° C. to about 65°C., such as at about 56° C., 58° C., 60° C., 62° C., or 64° C., for aperiod of time (e.g., about 30 minutes to about 6 hours, about 1 hour toabout 5 hours, about 1.5 hours to about 3 hours, or about 2 hours). Insome such embodiments, the fixed biological is baked uncovered in anoven. In some such embodiments, the baked fixed biological is calibratedto room temperature for a period of time (e.g., about 3 minutes to about30 minutes, about 5 minutes to about 20 minutes, or about 7 minutes).

In some such embodiments, for paraffin-embedded biological samples(e.g., FFPE samples), the sample can be de-paraffinized (e.g., toproduce a de-paraffinized fixed biological sample) and re-hydrated. Insome embodiments, de-paraffinizing comprises contacting the biologicalsample with xylene, ethanol, and water, or, sequentially contacting thebiological sample with xylene and an alcohol (e.g., ethanol) series suchas absolute ethanol, about 96% ethanol, and about 70% ethanol. In someembodiments, de-paraffinizing can include treating with xylene andethanol (e.g., absolute ethanol, about 96% ethanol, and or about 70%ethanol). In some embodiments, de-paraffinization can include,sequentially, treating with xylene (e.g., once, twice, or more times,each for about 5 minutes to about 15 minutes, such as about 10 minuteseach), treating with absolute ethanol (e.g., once, twice, or more times,each for about 1 minute to about 10 minutes, such as about 2 minute toabout 5 minutes, e.g., about 3 minutes each), treating with about 96%ethanol (e.g., once, twice, or more times, each for about 1 minute toabout 10 minutes, such as about 2 minute to about 5 minutes, e.g., about3 minutes each), and treating with about 70% ethanol (e.g., once, twice,or more times, each for about 1 minute to about 10 minutes, such asabout 2 minute to about 5 minutes, e.g., about 3 minutes each). In someembodiments, the sample can be treated with water for re-hydration(e.g., in nuclease free water (e.g., DEPC water)), e.g., once, twice, ormore times, each for about 5 seconds to about 1 minute, such as 10seconds to about 30 seconds, e.g., about 20 seconds each.

In some embodiments, a fixed biological sample is pretreated with one ormore pretreating reagents prior to delivery or application of ade-crosslinking agent (e.g., a catalyst disclosed herein). Pretreatmentcan include permeabilization of the biological sample, for example,using conditions milder than those typically used for extractinganalytes.

In some embodiments, a pretreating reagent can include a proteinase(e.g., collagenase). The proteinase can be present in any appropriateconcentration (e.g., about 0.005 to about 0.5 U/μL (e.g., about 0.01 toabout 0.5 U/μL, about 0.05 to about 0.5 U/μL, about 0.1 to about 0.5U/μL, about 0.1 to about 0.3 U/μL, or about 0.2 U/μL). In someembodiments, a proteinase can be pepsin, Proteinase K, or an ArcticZymesProteinase (an unspecific endopeptidase that can be inactivated afteruse). The proteinase can optionally be applied with a buffer, such asHank's Balanced Salt Solution (HBSS) buffer. In some embodiments, ifpepsin is used for permeabilization, a pretreating reagent can include aproteinase (e.g., a second proteinase or a proteinase other thanpepsin). In some embodiments, if Proteinase K is used forpermeabilization, a pretreating reagent may but does not need to includea proteinase.

In some embodiments, a pretreating reagent can include a detergent. Thedetergent can be present in any appropriate concentration (e.g., about0.05% to about 2% (v/v), about 0.1% to about 1% (v/v), about 0.1% (v/v),or about 0.5% (v/v)). In some embodiments, the detergent is a non-ionicdetergent. In some embodiments, the detergent comprises TRITON™ X-100.In some embodiments, the detergent is in a buffer. In some embodiments,the buffer comprises, for example,tris(hydroxymethyl)aminomethane-Ethylenediaminetetraacetic acid (TE),phosphate-buffered saline (PBS),2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), and/or2-(N-morpholino)ethanesulfonic acid (MES), with a pH of about 7.0 toabout 9.0 (e.g., about 7.5 to about 8.5, or about 8.0).

A pretreating reagent can be applied to the biological sample (e.g., acell or tissue sample such as a tissue section) in any number of ways.In some embodiments, a pretreating reagent is in solution or suspension.In some embodiments, the biological sample can be soaked in a solutionor suspension comprising a pretreating reagent. In some embodiments, apretreating reagent is sprayed onto the biological sample. In someembodiments, a pretreating reagent is supplied to the biological samplevia a microfluidic system (e.g., as a solution or suspension). In someembodiments, the biological sample is dipped into a solution orsuspension of a pretreating reagent, wherein excess solution orsuspension is removed from the biological sample. In some embodiments, apretreating reagent is delivered to the biological sample via ahydrogel, wherein the hydrogel is a repository for a pretreatmentreagent and is contacted with the biological sample.

The pretreatment can be applied to the biological sample (e.g., a cellor tissue sample such as a tissue section) for a time sufficient topermeabilize a biological sample to facilitate the de-crosslinking agent(e.g., a catalyst disclosed herein) penetrating the biological sample.In some embodiments, the pretreatment can be applied to the biologicalsample for between about 1 minute and about 60 minutes. In someembodiments, the pretreatment can be applied to the biological samplebetween about 1 minute and about 55 minutes, about 1 minute and about 50minutes, about 1 minute and about 45 minutes, about 1 minute and about40 minutes, about 1 minute and about 35 minutes, about 1 minute andabout 30 minutes, about 1 minute and about 25 minutes, about 1 minuteand about 20 minutes, about 5 minutes and about 60 minutes, about 10minutes and about 60 minutes, about 10 minutes and about 50 minutes,about 10 minutes and about 40 minutes, or about 10 minutes and about 30minutes. In some embodiments, the pretreatment can be applied to thebiological sample for about 20 minutes.

The biological sample (e.g., a cell or tissue sample such as a tissuesection) can be incubated during pretreatment. In some embodiments, thebiological sample can be incubated between about 30° C. and about 45° C.during pretreatment. In some embodiments, the biological sample can beat about 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C.,39° C., 40° C., 41° C., 42° C., 43° C., 44° C., or 45° C. duringpretreatment. In some embodiments, the biological sample can beincubated at about 37° C. during pretreatment.

In some embodiments, after drying, baking, de-paraffinization, and/orre-hydration, a fixed biological sample is not pretreated with one ormore pretreating reagents and prior to delivery or application of ade-crosslinking agent (e.g., a catalyst disclosed herein) to thebiological sample. In some embodiments, a de-paraffinized andre-hydrated biological is not pretreated by permeabilizing the sample(e.g., using a protease or detergent) prior to contacting the samplewith a de-crosslinking agent.

In some embodiments, the method comprises, prior to or after contactingthe biological sample with a de-crosslinking agent (e.g., a catalystdisclosed herein), a step of staining the biological sample and/orimaging the stained biological sample, optionally wherein the stainingcomprises the use of a histological stain and/or an immunological stain.A stain can be any appropriate stain, such as a histological stain(e.g., hematoxylin and eosin, H&E) or an immunological stain (e.g., animmunofluorescent stain), or any other stain described herein. In someembodiments, a stain may include an agent that interacts with an analyte(e.g., an endogenous analyte in a sample) indicative of cell featuresmay include an agent that interacts with an analyte (e.g., an endogenousanalyte in a sample) indicative of morphological features and/or cellfeatures, and the analyte may include intracellular analytes, such asproteins, protein modifications (e.g., phosphorylation status or otherpost-translational modifications), nuclear proteins, cellular membraneproteins, nuclear membrane proteins, or any combination thereof. In someinstances, the stain is a nucleic acid stain, an extracellular matrixstain, a cellular membrane stain, a nuclear membrane stain, acytological stain, and/or any combinations thereof.

Staining and/or imaging can be carried out before, during, and/or afterthe fixed biological sample (e.g., a fixed tissue section) isde-crosslinked using a de-crosslinking agent (e.g., a catalyst disclosedherein). In some embodiments, staining and/or imaging of the sampleafter de-crosslinking can be performed and the results can be comparedto those before de-crosslinking. Images of the sample can be used tomonitor cell or tissue morphology and/or sample detachment during sampleprocessing, including drying, baking, de-paraffinization, re-hydration,and/or de-crosslinking, as well as washing and/or permeabilization afterde-crosslinking.

C. De-Crosslinking Agents and Catalytic De-Crosslinking

Conditions for reversing the effects of fixing a biological sample tendto be harsh. See e.g., U.S. Pat. No. 7,919,280; US 2005/0014203; US2009/0202998A1; Masuda et al., “Analysis of chemical modification of RNAfrom formalin-fixed samples and optimization of molecular biologyapplications for such samples,” Nucleic Acids Research 27(22):4436-4443, (1999); Evers et al., “The effect of formaldehyde fixation onRNA: optimization of formaldehyde adduct removal,” Journal of MolecularDiagnostics 13(3): 282-288, (2011); and Beechem, “High-Plex spatiallyresolved RNA and protein detection using digital spatial profiling: Atechnology designed for immuno-oncology biomarker discovery andtranslational research,” Biomarkers for Immunotherapy of Cancer. Humana,New York, NY, 2020. 563-583, each of which is incorporated by referenceherein in its entirety. For example, treatment of PFA-treated tissuesamples can include heating to 60° C. to 70° C. in Tris buffer forseveral hours, and yet typically this removes only a fraction of thefixative-induced crosslinks. In another example, a 3.0% PFA-fixed tissuesamples were soaked in 20 mM Tris-HCl (pH 9.0) and then incubated for 2h at 60° C. for de-crosslinking (Nagaki et al., “De-crosslinking enablesvisualization of RNA—guided endonuclease—in situ labelling signals forDNA sequences in plant tissues,” Journal of Experimental Botany,71(6):1792-1800, (2020)). The harsh de-crosslinking treatment conditionscan result in permanent damage to biomolecules (e.g., nucleic acidanalytes and/or protein analytes, such as those described herein) in thesample.

In some embodiments, a de-crosslinking agent or un-fixing agent hereincan be a compound or composition that reverses fixation and/or removesthe crosslinks within or between biomolecules (e.g., analytes foranalytical methods, such as those described herein) in a sample causedby previous use of a fixation reagent. In some embodiments,de-crosslinking agents are compounds that act catalytically in removingcrosslinks in a fixed sample. In some embodiments, de-crosslinkingagents are compounds that act catalytically in removing aminalcrosslinks in a fixed sample. In some embodiments, de-crosslinkingagents can act on biological samples fixed with an aldehyde (e.g.,formaldehyde), an N-hydroxysuccinimide (NHS) ester, an imidoester, or acombination thereof.

In some embodiments, provided herein are catalysts that catalyzede-crosslinking of inter-molecular crosslinks and/or intra-molecularcrosslinks in the biological sample. In some embodiments, providedherein are catalysts that catalyze the cleavage of aminal bridges,thereby de-crosslinking the inter-molecular crosslinks and/orintra-molecular crosslinks.

In some embodiments, the catalyst is a water-soluble catalyst. In someembodiments, the catalyst is an organic molecule. In some embodiments,the catalyst is a transimination catalyst. In some embodiments, thecatalyst is a bifunctional transimination catalyst that accelerateshydrazone and oxime formation. In some embodiments, the catalystcatalyzes de-crosslinking of aminal crosslinks in the biological sample.In some embodiments, the catalyst catalyzes breakdown of hemi-aminaladducts and/or aminal adducts in the biological sample.

Aminal crosslinks (e.g., aminal bridges) can be catalytically reversedusing one or more organocatalyst. In some embodiments, in catalyticreversal of aminal crosslinks, a first C—N bond of the aminal bridge canbe broken in an acid-base reaction, and the second C—N bond of theaminal can be broken to generate repaired NH₂ groups on the first andsecond molecules. FIG. 2B shows aminal crosslinks can be catalyticallyreversed using a combination of acid catalysis and nucleophiliccatalysis.

In some embodiments, the catalyst is a compound of formula (I),

-   -   or a salt, zwitterion, or solvate thereof, wherein:    -   A is selected from the group consisting of —COOH, —P(═O)(OH)₂,        and —S(═O)₂OH;    -   X¹, X², X³, and X⁴ are each independently selected from the        group consisting of: CH, CR^(a), and N;    -   each occurrence of R^(a) is independently selected from the        group consisting of C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy,        —NO₂, —NR′R″, and —C(═O)NR′R″; and    -   each occurrence of R′ and R″ is independently selected from the        group consisting of H and C₁₋₆ alkyl which is optionally        substituted with

wherein n1 is an integer from 12 to 16.

In some embodiments of formula (I), it is provided that when A is—P(═O)(OH)₂ and X¹, X², and X⁴ are CH, then X³ is other than C—CH₃.

In some embodiments of formula (I), A is —COOH. In some embodiments offormula (I), A is —P(═O)(OH)₂. In some embodiments of formula (I), A is—S(═O)₂OH.

In some embodiments of Formula (I), X¹ is CH. In some embodiments ofFormula (I), X¹ is CR^(a). In certain of these embodiments, X¹ is C—CH₃.In some embodiments of Formula (I), X² is CH. In some embodiments ofFormula (I), X² is N. In some embodiments of Formula (I), X⁴ is CH. Insome embodiments of Formula (I), X⁴ is N.

In some embodiments of Formula (I), X³ is N. In some embodiments ofFormula (I), X³ is CH. In some embodiments of Formula (I), X³ is CR^(a).In certain of these embodiments, R^(a) is C₁₋₆ alkyl (e.g., methyl). Incertain embodiments, R^(a) is NO₂. In certain embodiments, R^(a) isNR′R″ (e.g., NH₂). In certain embodiments, R^(a) is C(═O)NR′R″. As anon-limiting example of the foregoing embodiments, R^(a) is

In some embodiments of Formula (I), X² and X⁴ are CH. In someembodiments of Formula (I), X¹, X², and X⁴ are CH. In certain of theseembodiments, X³ is CR^(a) (e.g., C—CH₃). In certain other embodiments,X³ is N. In certain of the foregoing embodiments (when X² and X⁴ are CH;or when X¹, X², and X⁴ are CH), A is —COOH or —P(═O)(OH)₂.

In some embodiments, the compound of Formula (I) is a compound ofFormula (IA):

or a salt, zwitterion, or solvate thereof.

In some embodiments of Formula (IA), A is —COOH. In some of theseembodiments of Formula (IA), R^(a) is C₁₋₆ alkyl. In certain of theseembodiments, R^(a) is C₁₋₃ alkyl. For example, in some embodiments,R^(a) is methyl. In other of these embodiments, R^(a) is methoxy. Inother of these embodiments, R^(a) is —NH₂. In other of theseembodiments, R^(a) is —N(CH₃)₂.

In some embodiments of Formula (IA), A is —P(═O)(OH)₂. In some of theseembodiments of Formula (IA), R^(a) is C₁₋₆ alkyl. In certain of theseembodiments, R^(a) is C₁₋₃ alkyl. For example, in some embodiments,R^(a) is methyl. In other of these embodiments, R^(a) is methoxy. Inother of these embodiments, R^(a) is —NH₂. In other of theseembodiments, R^(a) is —N(CH₃)₂.

In some embodiments of Formula (IA), A is —S(═O)₂OH. In some of theseembodiments of Formula (IA), R^(a) is C₁₋₆ alkyl. In certain of theseembodiments, R^(a) is C₁₋₃ alkyl. For example, in some embodiments,R^(a) is methyl. In other of these embodiments, R^(a) is methoxy. Inother of these embodiments, R^(a) is —NH₂. In other of theseembodiments, R^(a) is —N(CH₃)₂.

In some embodiments, the compound of Formula (I) is a compound ofFormula (IB):

or a salt, zwitterion, or solvate thereof, wherein: X³ is CH or N.

In some embodiments of Formula (IB), A is —P(═O)(OH)₂. In someembodiments of Formula (IB), X³ is N.

In some embodiments, the compound of Formula (I) is a compound ofFormula (IC):

or a salt, zwitterion, or solvate thereof, wherein R^(c) is an electronreleasing group. In some of these embodiments, the electron releasinggroup (R^(c)) is selected from the group consisting of alkyl, alkoxy,hydroxy, amino, alkylamino, dialkylamino, mercapto, alkylmercapto,silyloxy, aryloxy, and alkylthio. In some of these embodiments, theelectron releasing group is lower alkyl or lower alkoxy. In other ofthese embodiments, the electron releasing group is —NH₂. In still otherof these embodiments, the electron releasing group is —N(CH₃)₂.

In some embodiments, the compound of Formula (I) is a compound ofFormula (IC′):

or a salt, zwitterion, or solvate thereof, wherein R^(c) is an electronreleasing group. In some of these embodiments, the electron releasinggroup (R^(c)) is selected from the group consisting of alkyl, alkoxy,hydroxy, amino, alkylamino, dialkylamino, mercapto, alkylmercapto,silyloxy, aryloxy, and alkylthio. In some of these embodiments, theelectron releasing group is lower alkyl or lower alkoxy. In other ofthese embodiments, the electron releasing group is —NH₂. In still otherof these embodiments, the electron releasing group is —N(CH₃)₂.

In some embodiments, the sample is contacted with a compound (e.g., in asolution or suspension) for catalytic de-crosslinking selected from thegroup consisting of 2-amino-5-methylbenzoic acid, 2-amino-5-nitrobenzoicacid, (2-amino-5-methylphenyl)phosphonic acid,2-amino-5-methylbenzenesulfonic acid, 2,5-diaminobenzenesulfonic acid,2-amino-3,5-dimethylbenzenesulfonic acid,(2-amino-5-nitrophenyl)phosphonic acid, (4-aminopyridin-3-yl)phosphonicacid, (3-aminopyridin-2-yl)phosphonic acid,(5-aminopyrimidin-4-yl)phosphonic acid,(2-amino-5-{[2-poly-ethoxy]ethyl}carbamoyl)phenyl)phosphonic acid,4-aminonicotinic acid, 3-aminoisonicotinic acid, 2-aminonicotinic acid,and (2-aminophenyl)phosphonic acid. In some embodiments, the sample iscontacted with Compound 1 (2-amino-5-methylbenzoic acid) in a solutionor suspension for catalytic de-crosslinking. In some embodiments, thesample is contacted with Compound 8 ((4-aminopyridin-3-yl)phosphonicacid) in a solution or suspension for catalytic de-crosslinking. In someembodiments, the sample is contacted with Compound 15((2-aminophenyl)phosphonic acid) in a solution or suspension forcatalytic de-crosslinking.

In some embodiments, the catalyst of formula (I) is selected from thegroup consisting of:

Compound No. Compound Structure    1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

or a salt, zwitterion, or solvate thereof.

In some embodiments, the catalyst comprises

or a salt zwitterion, or solvate thereof. In some embodiments, thecatalyst comprises

or a salt, zwitterion, or solvate thereof. In some embodiments, thecatalyst comprises

or a salt, zwitterion, or solvate thereof. In some embodiments, thecatalyst comprises

or a salt, zwitterion, or solvate thereof. In some embodiments, thecatalyst comprises

or a salt, zwitterion, or solvate thereof. In some embodiments, thecatalyst comprises

or a salt, zwitterion, or solvate thereof. In some embodiments, thecatalyst comprises

or a salt, zwitterion, or solvate thereof. In some embodiments, thecatalyst comprise

or a salt, zwitterion, or solvate thereof. In some embodiments, thecatalyst comprises

or a salt, zwitterion, or solvate thereof. In some embodiments, thecatalyst comprises

or a salt, zwitterion, or solvate thereof. In some embodiments, thecatalyst comprises

or a zwitterion, or solvate thereof. In some embodiments, the catalystcomprises

or a salt, zwitterion, or solvate thereof. In some embodiments, thecatalyst comprises

or a salt, zwitterion, or solvate thereof. In some embodiments, thecatalyst comprises

or a salt, zwitterion, or solvate thereof. In some embodiments, thecatalyst comprises

or a salt, zwitterion, or solvate thereof.

In some embodiments, a compound disclosed herein catalytically breaksdown the aminal and hemi-aminal adducts that form in RNA treated withformaldehyde, and is compatible with many RNA extraction and detectionconditions. Exemplary compounds include those described in Karmakar etal., “Organocatalytic removal of formaldehyde adducts from RNA and DNAbases,” Nature Chemistry, 7: 752-758 (2015); US 2017/0283860; and US2019/0135774, each of which is incorporated by reference herein in itsentirety.

In some embodiments, the catalyst is a compound of formula (II):

-   -   or a salt, zwitterion, or solvate thereof, wherein:    -   L¹ is selected from the group consisting of —O—, —N(H)—, —N(C₁₋₃        alkyl), —N(CH₂CH₂O)₁₋₁₀—CH₃—, —S(O)₀₋₂—, —CH₂—, and a bond;    -   R¹ is selected from the group consisting of: H; C₁₋₆ alkyl; C₁₋₆        haloalkyl; C₆₋₁₀ aryl optionally substituted with 1-4 R^(b); and        5- to 10-membered heteroaryl, wherein 1-4 ring atoms are        heteroatoms each independently selected from the group        consisting of: N, N(H), N(C₁₋₃ alkyl), O, and S, wherein the        heteroaryl is optionally substituted with 1-4 independently        selected R^(b); and    -   each R^(b) is independently selected from the group consisting        of: halo, cyano, —OH, —NH₂, —NH(C₁₋₃ alkyl), —N(C₁₋₃ alkyl)₂,        C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₄ alkoxy, and C₁₋₄ haloalkoxy.

In some embodiments of formula (II), -L¹-R¹ and the —COOH group are cisto one another. In some embodiments of formula (II), -L¹-R¹ and the—COOH group are trans to one another.

In some embodiments of formula (II), the catalyst is a compound offormula (II-a):

or a salt, zwitterion, or solvate thereof.

In some embodiments of formula (II), the catalyst is a compound offormula (II-a1):

or a salt, zwitterion, or solvate thereof.

In some embodiments of formula (II), the catalyst is a compound offormula (II-a2):

or a salt, zwitterion, or solvate thereof.

In some embodiments of formula (II), the catalyst is a compound offormula (II-b):

or a salt, zwitterion, or solvate thereof.

In some embodiments of formula (II), the catalyst is a compound offormula (II-b1):

or a salt, zwitterion, or solvate thereof.

In some embodiments of formula (II), the catalyst is a compound offormula (II-b2):

or a salt, zwitterion, or solvate thereof.

In some embodiments of formula (II), (II-a), (II-a1), (II-a2), (II-b),(II-b1), or (II-b2), L¹ is —O—. In some embodiments of formula (II),(II-a), (II-a1), (II-a2), (II-b), (II-b1), or (II-b2), L¹ is —N(H)— or—N(C₁₋₃ alkyl)-. In certain of these embodiments, L¹ is —N(H)—.

In some embodiments of formula (II), (II-a), (II-a1), (II-a2), (II-b),(II-b1), or (II-b2), R¹ is H.

In some embodiments of formula (II), (II-a), (II-a1), (II-a2), (II-b),(II-b1), or (II-b2), R¹ is a heteroaryl containing 5-10 ring atoms,wherein 1-4 ring atoms are heteroatoms each independently selected fromthe group consisting of: N, N(H), N(C₁₋₃ alkyl), O, and S; and whereinthe heteroaryl is optionally substituted with 1-4 independently selectedR^(b).

In certain of these embodiments, R¹ is a heteroaryl containing 5-6 ringatoms, wherein 1-4 ring atoms are heteroatoms each independentlyselected from the group consisting of: N, N(H), N(C₁₋₃ alkyl), O, and S;and wherein the heteroaryl is optionally substituted with 1-2independently selected R^(b).

In certain of the foregoing embodiments, R¹ is a heteroaryl containing 6ring atoms, wherein 1-2 ring atoms are ring nitrogen atoms, and whereinthe heteroaryl is optionally substituted with 1-2 independently selectedR^(b).

As a non-limiting example of the foregoing embodiments, R¹ can bepyridyl, which is optionally substituted with 1-2 independently selectedR^(b). For example, R¹ can be 3-pyridyl, which is optionally substitutedwith 1-2 independently selected R^(b) (e.g., unsubstituted 3-pyridyl,3-pyridyl substituted with one R^(b), or 3-pyridyl substituted with twoR^(b)). As another non-limiting example, R¹ can be 4-pyridyl which isoptionally substituted with 1-2 R^(b) (e.g., unsubstituted 4-pyridyl,4-pyridyl substituted with one R^(b), or 4-pyridyl substituted with twoR).

In some embodiments of formula (II), the catalyst is a compound offormula (II-a1); L¹ is —O—; and R¹ is heteroaryl containing 6 ringatoms, wherein 1-2 ring atoms are ring nitrogen atoms, and wherein theheteroaryl is optionally substituted with 1-2 independently selectedR^(b). In certain of these embodiments, R¹ is pyridyl which isoptionally substituted with 1-2 independently selected R^(b). Forexample, R¹ can be 3-pyridyl which is optionally substituted with 1-2independently selected R^(b) (e.g., unsubstituted 3-pyridyl). As anothernon-limiting example, R¹ can be 4-pyridyl which is optionallysubstituted with 1-2 R^(b) (e.g., unsubstituted 4-pyridyl).

In some embodiments of formula (II), the catalyst is a compound offormula (II-a1); L¹ is —O—, —N(H)—, or —N(C₁₋₃ alkyl)-; and R¹ is H.

In some embodiments, the catalyst is selected from the group consistingof (2S,4R)-4-hydroxyproline, (2R,4S)-4-hydroxyproline,(2S,4S)-4-hydroxyproline, (2R,4R)-4-hydroxyproline,(2S,4R)-4-aminoproline, (2R,4S)-4-aminoproline, (2S,4S)-4-aminoproline,and (2R,4R)-4-aminoproline.

In some embodiments, the catalyst of formula (II) is selected from thegroup consisting of.

Compound No. Compound Structure   18

19

20

21

22

23

24

25

26

27

or a salt, zwitterion, or solvate thereof.

In some embodiments, the catalyst comprises

or a salt, zwitterion, or solvate thereof. In some embodiments, thecatalyst comprises

or a salt, zwitterion, or solvate thereof. In some embodiments, thecatalyst comprises

or a salt, zwitterion, or solvate thereof. In some embodiments, thecatalyst comprises

or a salt, zwitterion, or solvate thereof. In some embodiments, thecatalyst comprises

or a salt, zwitterion, or solvate thereof. In some embodiments, thecatalyst comprises

or a salt, zwitterion, or solvate thereof.

In some embodiments, the catalyst is selected from the group consistingof:

Compound No. Compound Structure    1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

or a salt, zwitterion, or solvate thereof.

A catalyst can be contacted with (e.g., applied to) a biological sampleat any appropriate concentration. An appropriate concentration maydepend on factors such as tissue type, fixation reagent used, and degreeof crosslinking in the biological sample. In some embodiments, acatalyst can be contacted with (e.g., applied to) a biological sample ina solution or suspension with a concentration of about 5 mM to about 500mM (e.g., about 10 mM to about 100 mM, about 10 mM to about 200 mM,about 10 mM to about 300 mM, about 10 mM to about 400 mM, about 100 mMto about 200 mM, about 100 mM to about 300 mM, about 100 mM to about 400mM, about 100 mM to about 500 mM, about 200 mM to about 300 mM, about200 mM to about 400 mM, about 200 mM to about 500 mM, about 300 mM toabout 400 mM, about 300 mM to about 500 mM, or about 400 mM to about 500mM). In some embodiments, a catalyst can be contacted with (e.g.,applied to) a biological sample in a solution or suspension with aconcentration of about 10 mM to about 100 mM (e.g., about 10 mM to about20 mM, about 10 mM to about 30 mM, about 10 mM to about 40 mM, about 10mM to about 50 mM, about 10 mM to about 60 mM, about 10 mM to about 70mM, about 10 mM to about 80 mM, about 10 mM to about 90 mM, about 20 mMto about 30 mM, about 20 mM to about 40 mM, about 20 mM to about 50 mM,about 20 mM to about 60 mM, about 20 mM to about 70 mM, about 20 mM toabout 80 mM, about 20 mM to about 90 mM, about 20 mM to about 100 mM,about 30 mM to about 40 mM, about 30 mM to about 50 mM, about 30 mM toabout 60 mM, about 30 mM to about 70 mM, about 30 mM to about 80 mM,about 30 mM to about 90 mM, about 30 mM to about 100 mM, about 40 mM toabout 50 mM, about 40 mM to about 60 mM, about 40 mM to about 70 mM,about 40 mM to about 80 mM, about 40 mM to about 90 mM, about 40 mM toabout 100 mM, about 50 mM to about 60 mM, about 50 mM to about 70 mM,about 50 mM to about 80 mM, about 50 mM to about 90 mM, about 50 mM toabout 100 mM, about 60 mM to about 70 mM, about 60 mM to about 80 mM,about 60 mM to about 90 mM, about 60 mM to about 100 mM, about 70 mM toabout 80 mM, about 70 mM to about 90 mM, about 70 mM to about 100 mM,about 80 mM to about 90 mM, about 80 mM to about 100 mM, or about 90 mMto about 100 mM) of the catalyst. In some embodiments, a catalyst can becontacted with (e.g., applied to) a biological sample in a solution orsuspension with a concentration of about 30 mM to about 70 mM of thecatalyst. In some embodiments, a catalyst can be contacted with (e.g.,applied to) a biological sample in a solution or suspension with aconcentration of about 40 mM to about 60 mM of the catalyst. In someembodiments, a catalyst can be contacted with (e.g., applied to) abiological sample in a solution or suspension with a concentration ofabout 50 mM of the catalyst.

In some embodiments, the catalyst is contacted with the biologicalsample at a concentration between about 5 mM and about 500 mM. In someembodiments, the catalyst is contacted with the biological sample at aconcentration between about 10 mM and about 400 mM. In some embodiments,the catalyst is contacted with the biological sample at a concentrationbetween about 50 mM and about 300 nM. In some embodiments, the catalystis contacted with the biological sample at a concentration between about75 mM and about 250 mM In some embodiments, the catalyst is contactedwith the biological sample at a concentration between about 100 mM andabout 200 mM, such as about 100 mM, about 110 mM, about 120 mM, about130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about180 mM, about 190 mM, or about 200 mM.

The catalyst can be contacted to the biological sample for a timesufficient to de-crosslink some or all of the crosslinked nucleic acidsand/or proteins in the biological sample (e.g., a cell or tissue samplesuch as a tissue section). In some embodiments, the catalyst can becontacted to the biological sample for between 1 minute and 1 day (e.g.,between 1 minute and 1 hour, 1 minute and 2 hours, 1 minute and 4 hours,1 minute and 6 hours, 1 minute and 12 hours, 1 minute and 18 hours, 1hour and 2 hours, 1 hour and 4 hours, 1 hour and 6 hours, 1 hour and 12hours, 1 hour and 18 hours, 1 hour and 1 day, 2 hours and 4 hours, 2hours and 6 hours, 2 hours and 12 hours, 2 hours and 18 hours, 2 hoursand 1 day, 4 hours and 6 hours, 4 hours and 12 hours, 4 hours and 18hours, 4 hours and 1 day, 6 hours and 12 hours, 6 hours and 18 hours, 6hours and 1 day, 12 hours and 18 hours, 12 hours and 1 day, or 18 hoursand 1 day). In some embodiments, the catalyst is contacted with thebiological sample for about 1 minute to about 150 minutes. In someembodiments, the catalyst is contacted with the biological sample forabout 5 minute to about 100 minutes. In some embodiments, the catalystis contacted with the biological sample for about 10 minute to about 50minutes. In some embodiments, the catalyst is contacted with thebiological sample for about 15 minute to about 30 minutes.

The catalyst can be contacted to the biological sample at a temperaturesufficient to de-crosslink some or all of the crosslinked nucleic acidsand/or proteins in the biological sample (e.g., a cell or tissue samplesuch as a tissue section). In some embodiments, the catalyst iscontacted with the biological sample at a temperature between about 5°C. and about 100° C. In some embodiments, the catalyst is contacted withthe biological sample at a temperature between about 50° C. and about95° C. In some embodiments, the catalyst is contacted with thebiological sample at a temperature between about 60° C. and about 90°C., such as about 60° C., about 65° C., about 70° C., about 75° C.,about 80° C., about 85° C., or about 90° C.

A biological sample (e.g., a cell or tissue sample such as a tissuesection) can be incubated while the catalyst is contacted with (e.g.,applied to) the biological sample. In some embodiments, the biologicalsample can be incubated between about 25° C. and about 100° C. In someembodiments, the biological sample can be incubated between about 25° C.and about 40° C., about 37° C. and about 60° C., about 45° C. and about95° C., about 50° C. and about 90° C., about 55° C. and about 85° C.,about 60° C. and about 85° C., about 75° C. and about 85° C. In someembodiments, the biological sample can be incubated at about 80° C.

In some embodiments, an incubation temperature and a contact time can berelated. Without being bound by any particular theory, it is believedthat if a higher temperature is used, a shorter contact time may besufficient (e.g., 70° C. to 80° C. for 30 minutes), while if a lowertemperature is used, a longer contact time may be beneficial (e.g., 37°C. for 1 day). However, in some cases, both a low temperature and ashorter contact time may be sufficient (e.g., 20° C. to 28° C. for 90minutes). In some embodiments, the catalyst can be contacted to thebiological sample for between 1 hour and 120 minutes (e.g., between 1minute and 110 minutes, 1 minute and 100 minutes, 1 minute and 90minutes, 1 minute and 80 minutes, 1 minute and 70 minutes, 10 minutesand 120 minutes, 20 minutes and 120 minutes, 30 minutes and 120 minutes,40 minutes and 120 minutes, 50 minutes and 120 minutes. In someembodiments, the catalyst agent can be applied to the biological samplefor about 10 minutes, about 20, 30, 40, 50, 60, 70, 80, 90, 110, orabout 120 minutes, and at a temperature between about 70° C. and about95° C., such as about 70° C., about 75° C., about 80° C., about 85° C.,about 90° C., or 95° C. In some embodiments, the catalyst can becontacted to the biological sample for approximately 30 minutes at atemperature between about 75° C. and about 85° C., such as about 80° C.

A catalyst can be delivered to a biological sample using any appropriatemethod. In some embodiments, a catalyst can be delivered as a solutionor a suspension. In some embodiments, a catalyst can be delivered as asolution or a suspension in a buffer. In some embodiments, the buffer iscitrate, tris(hydroxymethyl)aminomethane (Tris), Tris-EDTA,phosphate-buffered saline (PBS),2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES),2-(N-morpholino)ethanesulfonic acid (MES), or a combination thereof. Insome embodiments, the buffer is Tris. In some embodiments, the buffercomprises Tris and a chelating agent, optionally wherein the chelatingagent is ethylenediaminetetraacetic acid (EDTA) and the buffer isTris-EDTA (TE) In some embodiments, the buffer comprises citrate. Insome embodiments, the buffer comprises citrate and dimethyl sulfoxide(DMSO). In some embodiments, the buffer comprises 1%-5% (v/v) DMSO. Insome embodiments, the buffer comprises 2% (v/v) DMSO. In someembodiments, the buffer comprises citrate but no DMSO A buffer can haveany appropriate concentration. For example, in some embodiments, abuffer can have a concentration of about 5 mM to about 60 mM (e.g.,about 10 mM to about 50 mM, about 20 mM to about 40 mM, or about 30 mM).In some embodiments, the catalyst is formulated with DMSO and combinedwith the buffer (e.g., a citrate buffer, a PBS buffer, or a TE buffer)or before contacting the biological sample.

In some embodiments, the buffer is present in the solution or suspensionat a concentration between about 5 mM and about 300 mM. In someembodiments, the buffer is present in the solution or suspension at aconcentration between about 10 mM and about. 250 mM, such as betweenabout 100 mM and about 200 mM, such as about 100 mM, about 110 mM, about120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about170 mM, about 180 mM, about 190 mM, or about 200 mM.

In some embodiments, the solution or suspension has a pH between about 4and about 10. In some embodiments, the solution or suspension has a pHbetween about 6 and about 9. In some embodiments, the solution orsuspension has a pH between about 6.5 and about 8, such as between about6.8 and about 7.4. In some embodiments, the buffer is present at aconcentration between about 100 mM and about 200 mM in the solution orsuspension having a pH between about 6.5 and about 8, such as pH 6.6, pH6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, or pH 7.3, pH 7.4, pH 7.5,pH 7.6, pH 7.7, pH 7.8, or pH 7.9.

In some embodiments, the compound in the solution or suspension canexist in various forms, e.g., depending on the pH of the solution orsuspension, and one or more of the forms can function as a catalyst tode-crosslink molecular crosslinks in the sample. Examples ofpH-dependent acid catalysis are shown in FIGS. 3A-3C. For instance, inFIG. 3B, Compound 1 can exist in two forms—compound A and compound B—ina solution at pH 6.8, but only compound B with —COOH may catalyze ade-crosslinking reaction for reversal of aminal crosslinks. At pH 9,Compound 1 exists mostly as compound A, which is a deprotonated formthat does not catalyze the de-crosslinking reaction. FIG. 3C showsphosphonic acid catalysts may perform well at pH 6.8 since —OH of thephosphonic acid exists in solution to catalyze a de-crosslinkingreaction exemplified in FIG. 2C.

Exemplary reagents for inclusion in the solution or suspension describedherein may include, but are not limited to, water, various non-ionicdetergents, saline-sodium citrate (SSC), sodium phosphate, phosphatebuffered saline (PBS), sodium dodecyl sulfate (SDS), urea, proteinase(e.g., proteinase K), bovine serum albumin (BSA),ethylenediaminetetracetic acid (EDTA), a sarkosyl compound (e.g., sodiumlauroyl sarcosinate; sarkosyl, ammonium salt; or sarkosyl, potassiumsalt), tris(hydroxymethyl)aminomethane (tris), tris-HCl (trishydrochloride), 3-morpholinopropane-1-sulfonic acid (MOPS), TAE buffer(tris EDTA), TBS buffer (tris buffered saline), bis-tris methane,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES buffer),dimethyl sulfoxide (DMSO), quaternary ammonium salts (e.g.,tetramethylammonium chloride (TMAC)), trimethybenzylammonium chloride(TMBAC), tetraethylphosphonium chloride (TEPC), triethylbenzylammoniumchloride (TEBAC), tetra-n-propylammonium chloride (TPAC),tri-n-butylbenzylammonium chloride (TBBAC), tetra-n-butylphosphoniumchloride (TBPC), (3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate) (3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate) (CHAPS detergent), and cholinedihydrogen phosphate (choline DHP). The solution or suspension mayinclude a zwitterionic catalyst and/or a zwitterionic detergent.

In some embodiments, the solution or suspension comprises sodium dodecylsulfate (SDS), urea, and/or a proteinase, optionally wherein theproteinase is proteinase K. In some embodiments, the solution orsuspension comprises SDS and proteinase K. In some embodiments, thesolution or suspension comprises urea and proteinase K. In someembodiments, the urea concentration is between about 0.01 M and about 1M, such as 0.01 M, 0.02 M, 0.05 M, 0.1 M, 0.2 M, 0.5 M, 0.75 M, or 1 M,or any concentration in between the aforementioned values. In someembodiments, the proteinase K concentration is between about 0.1 μg/mLand about 2 μg/mL, such as 0.1 μg/mL, 0.2 μg/mL, 0.5 μg/mL, 0.75 μg/mL,1 μg/mL, 1.25 μg/mL, 1.5 μg/mL, 1.75 μg/mL, or 2 μg/mL, or anyconcentration in between the aforementioned values. In some embodiments,the SDS concentration (w/v) is between about 0.01% and about 1%, such as0.05%, 0.1%, 0.2%, 0.5%, 0.75%, or 1%, or any concentration in betweenthe aforementioned values. In some embodiments, the solution orsuspension comprises 0.05% SDS, 0.2% SDS, 0.5% SDS, 0.05 M urea, 0.5 Murea, 0.2 μg/ml proteinase K, 0.5 μg/ml proteinase K, 1 μg/ml proteinaseK, or any combination thereof. In some embodiments, using SDS, urea,and/or proteinase K in de-crosslinking conditions increases punctabrightness, object counts (e.g., puncta number per unit area), and/orsignal-to-noise ratio.

In some embodiments, disclosed herein is a catalyst (e.g., a compound offormula (I) or (II) disclosed herein) in a solution or suspensioncomprising phosphate buffered saline (PBS), e.g., a PBS buffer solutionhaving a pH between about 6.5 and about 8, such as pH 6.6, pH 6.7, pH6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, or pH 7.3, pH 7.4, pH 7.5, pH 7.6,pH 7.7, pH 7.8, or pH 7.9. In some embodiments, disclosed herein is acatalyst (e.g., a compound of formula (I) or (II) disclosed herein) in aPBS buffer solution having a pH of about 7.4. In some embodiments,disclosed herein is a compound of formula (I), such as2-amino-5-methylbenzoic acid, (2-aminophenyl)phosphonic acid, and/or(4-aminopyridin-3-yl)phosphonic acid, as well as a PBS buffer solutionhaving a pH of about 7.4 comprising the compound of formula (I).

In some embodiments, disclosed herein is a catalyst (e.g., a compound offormula (I) or (II) disclosed herein) in a solution or suspensioncomprising citrate, e.g., a citrate solution having a pH between about 5and about 8, such as about pH 5.5, about pH 6.0, about pH 6.5, about pH7.0, or about pH 7.5 In some embodiments, disclosed herein is a catalyst(e.g., a compound of formula (I) or (II) disclosed herein) in a citratebuffer solution having a pH of about 6.0. In some embodiments, disclosedherein is a compound of formula (I), such as 2-amino-5-methylbenzoicacid, (2-aminophenyl)phosphonic acid, and/or(4-aminopyridin-3-yl)phosphonic acid, as well as a citrate buffersolution having a pH of about 6.0 comprising the compound of formula(I).

In some embodiments, disclosed herein is a catalyst (e.g., a compound offormula (I) or (II) disclosed herein) in a solution or suspensioncomprising Tris, e.g., a Tris-EDTA solution having a pH between about 8and about 10, such as about pH 8.5, about pH 9.0, about pH 9.5, or aboutpH 10.0. In some embodiments, disclosed herein is a catalyst (e.g., acompound of formula (I) or (II) disclosed herein) in a Tris-EDTA buffersolution having a pH of about 9.0. In some embodiments, disclosed hereinis a compound of formula (I), such as 2-amino-5-methylbenzoic acid,(2-aminophenyl)phosphonic acid, and/or (4-aminopyridin-3-yl)phosphonicacid, as well as a Tris-EDTA buffer solution having a pH of about 9.0comprising the compound of formula (I).

A catalyst can be contacted with (e.g., applied to) the biologicalsample (e.g., a cell or tissue sample such as a tissue section) in anynumber of ways. In some embodiments, a catalyst is in solution or asuspension. In some embodiments, the biological sample is soaked in asolution or suspension comprising the catalyst. In some embodiments, thecatalyst is sprayed onto the biological sample, e.g., as a solution orsuspension. In some embodiments, the catalyst is supplied to thebiological sample via a microfluidic system (e.g., as a solution orsuspension). In some embodiments, a catalyst is pipetted or otherwisealiquoted onto the biological sample. In some embodiments, thebiological sample is dipped into a solution or suspension of a catalyst,wherein excess solution or suspension is removed from the biologicalsample. In some embodiments, a catalyst can be delivered to thebiological sample via a hydrogel, wherein the hydrogel is contacted withthe biological sample. Application of a catalyst can occur in othersuitable ways.

In some embodiments, a composition comprising a catalyst or compounddisclosed herein can be used in combination with one or more otherde-crosslinking and/or antigen retrieving agents including enzymes suchas proteases. Exemplary proteases include a proteinase (e.g.,collagenase), which can be present in any appropriate concentration(e.g., about 0.005 to about 0.5 U/μL (e.g., about 0.01 to about 0.5U/μL, about 0.05 to about 0.5 U/μL, about 0.1 to about 0.5 U/μL, about0.1 to about 0.3 U/μL, or about 0.2 U/μL). In some embodiments, aproteinase can be pepsin, Proteinase K, or an ArcticZymes Proteinase (anunspecific endopeptidase that can be inactivated after use). Theproteinase can optionally be applied with a buffer, such as Hank'sBalanced Salt Solution (HBSS) buffer.

In some embodiments, a composition comprising a catalyst or compounddisclosed herein can be used in combination with one or more crowdingagents, e.g., to mitigate potential RNA loss during de-crosslinking.Crowding agents are typically high-molecular weight, high valencypolymers that may be charged. For example, crowding agents may bepolymers such as dextran sulfate, polyacrylic acid, polyvinylsulfonicacid, and alginate. Optionally, crowding agents may be polymers similarto dextran sulfate. In some embodiments, compounds that can function ascrowding agents, but have some property of molecular programmability maybe used. For example, some polymers that can function as crowding agentscan be used and subsequently the charged group can be cleaved off orneutralized. This can convert the compound into a neutral polymer likePEG, which actually enhances the efficiency of enzymatic reactions. Insome embodiments, polymers can function as a crowding agent, and then bespecifically degraded into small monomers and can be easily washed fromthe sample. Examples of programmable polyions or polyelectrolytes forenzyme-compatible enhancement of nucleic acid hybridization kinetics(e.g., for hybridization of nucleic acid probes to molecules in thesample after de-crosslinking) include polycondensation reactions ofCys(Lys)_(n)Cys, polymers such as PEG (PEG20k MW), PVA, or PAA, whichmay be subsequently modified via a cleavable linker to include chemicalgroups conferring ionic charge, or polymers formed from monomersincluding cleavable linkages, such that the polymer may be degradedsubsequent to functioning as a crowding agent.

D. Preparing De-Crosslinked Samples for In Situ Analysis

In some embodiments, after catalytic de-crosslinking, a biologicalsample (e.g., tissue section) is permeabilized (e.g., undergoespermeabilization). In some embodiments, the sample is permeabilizedafter delivery to or application of a de-crosslinking agent. In someembodiments, the permeabilization comprises harsher conditions than theoptional pretreatment step. In some embodiments, the permeabilizationcomprises applying one or more permeabilization reagents to thebiological sample. In some embodiments, a permeabilization reagent cancomprise a protease. In some embodiments, the protease comprises pepsin.In some embodiments, the protease comprises proteinase K. In someembodiments, the protease comprises ArcticZyme Proteinase. In someembodiments, the protease is provided in a solution of hydrochloricacid. In some embodiments, after catalytic de-crosslinking, a biologicalsample (e.g., tissue section) is contacted with a protease, e.g., washedusing a solution containing a protease, prior to contacting thebiological sample with a labelling agent (e.g., a probe or probe set)that directly or indirectly binds to an analyte at a location in thebiological sample. In some examples, provided is a method comprisingcontacting an intact biological sample with a labelling agent thatdirectly or indirectly binds to an analyte at a location in thebiological sample.

The permeabilization reagent(s) can be applied to biological sample(e.g., tissue section) in any number of ways. In some embodiments, thepermeabilization reagents can be in solution or suspension. In someembodiments, the sample is soaked in a solution or suspension of thepermeabilization reagent(s). In some embodiments, the permeabilizationreagents are sprayed onto the sample (e.g., as a solution orsuspension). In some embodiments, the permeabilization reagents aresupplied to the sample via a microfluidic system (e.g., as a solution orsuspension). In some embodiments, the sample can be dipped into asolution or suspension comprising the permeabilization reagent(s),wherein excess permeabilization reagent is removed from the sample. Insome embodiments, the permeabilization reagent(s) are delivered to thesample via a hydrogel, wherein the hydrogel is contacted with thesample.

The permeabilization reagent(s) can be contacted to the biologicalsample (e.g., tissue section) for a time sufficient to permeabilize thesample. In some embodiments, the permeabilization reagent(s) can becontacted to the sample for between 1 minute and 120 minutes. In someembodiments, the permeabilization reagent(s) can be applied to thesample between about 1 minute and 90 minutes, 1 minute and 80 minutes, 1minute and 70 minutes, 1 minute and 60 minutes, 1 minute and about 55minutes, about 1 minute and about 50 minutes, about 1 minute and about45 minutes, about 1 minute and about 40 minutes, about 1 minute andabout 35 minutes, about 1 minute and about 30 minutes, about 1 minuteabout 5 minutes and about 60 minutes, about 10 minutes and about 60minutes, about 10 minutes and about 55 minutes, about 10 minutes andabout 50 minutes, about 10 minutes and about 45 minutes, about 10minutes and about 40 minutes, about 15 minutes and about 45 minutes,about 20 minutes and about 40 minutes, about 25 minutes and about 35minutes. In some embodiments, the permeabilization reagent(s) can becontacted to the sample for approximately 30 minutes.

The biological sample (e.g., tissue section) can be incubated with thepermeabilization reagent(s). In some embodiments, the sample can beincubated between about 16° C. and about 56° C. (e.g., between about 30°C. and 45° C., or between about 35° C. and about 40° C.). In someembodiments, the biological sample can be at about 16° C., 18° C., 20°C., 22° C., 24° C., 26° C., 28° C., 30° C., 31° C., 32° C., 33° C., 34°C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43°C., 44° C., 45° C., 46° C., 48° C., 50° C., 52° C., 54° C., or 56° C. Insome embodiments, the sample can be incubated at about 37° C.

In some embodiments, the method comprises a step of permeabilizing thebiological sample before, during, and/or after contacting the biologicalsample with the catalyst for catalytic de-crosslinking. In someembodiments, the method comprises incubating and/or washing a fixedbiological sample in a buffer comprising one or more detergents, such asa Tween detergent (e.g., Tween™ 20), before and/or during catalyticde-crosslinking. In some embodiments, the method comprises incubatingand/or washing a catalytically de-crosslinked biological sample in abuffer comprising one or more detergents, such as a Tween detergent(e.g., Tween™ 20.

In some embodiments, the method does not comprise migrating molecules(e.g., analytes, labelling agents, nucleic acid probes, etc., orproducts generated in situ in the sample) outside of the permeabilizedbiological sample. In some embodiments, the method does not comprisemigrating molecules (e.g., analytes, labelling agents, nucleic acidprobes, etc., or products generated in situ in the sample) towards thesubstrate, optionally wherein the migration is passive migration oractive migration. In some embodiments, the method does not comprisecapturing molecules (e.g., analytes, labelling agents, nucleic acidprobes, etc., or products generated in situ in the sample) by a captureagent immobilized on the substrate.

In some embodiments, after catalytic de-crosslinking, a biologicalsample (e.g., tissue section) can be incubated and/or washed with asolution containing a detergent in any appropriate concentration (e.g.,about 0.05% to about 2% (v/v), about 0.1% to about 1% (v/v), about 0.1%(v/v), or about 0.5% (v/v)). In some embodiments, the detergent is anon-ionic detergent. In some embodiments, the detergent comprisesTriton™ X-100, Triton™ X-200, Tween™ 20, Tween™ 80, N-lauroyl sarcosine,sodium dodecyl sulfate (SDS), dodecyldimethylphosphine oxide, sorbitanmonopalmitate, decylhexaglycol, 4-nonylphenylpolyethylene glycol, CAHPS,IGEPAL CA-630, Sulfobetain-10, Sulfobetain-16, urea, or a combinationthereof. In some embodiments, urea solubilizes and denatures proteins bydisrupting noncovalent bonds. In some embodiments, the buffer comprises,for example, tris(hydroxymethyl)aminomethane-Ethylenediaminetetraaceticacid (TE), phosphate-buffered saline (PBS),2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), and/or2-(N-morpholino)ethanesulfonic acid (MES), with a pH of about 7.0 toabout 9.0 (e.g., about 7.5 to about 8.5, or about 8.0). In someembodiments, the detergent is in a buffer such as PBS. In someembodiments, the buffer is PBS with a Tween detergent (PBST) In someembodiments, the method comprises, after contacting the biologicalsample with the catalyst, a step of washing the biological sample,optionally wherein the biological sample is washed in PBST, e.g., forthree times for 1 minute each.

In some embodiments, the method comprises, after contacting thebiological sample with the catalyst, a step of staining the biologicalsample and imaging the stained biological sample prior to containing thesample with a labelling agent that directly or indirectly binds to ananalyte at a location in the biological sample, e.g., for analytedetection using nucleic acid probes for in situ hybridization (e.g.,sequential hybridization in sequential rounds of probe hybridization anddetection) and/or using labelled antibodies for protein detection (e.g.,described in Section III-B and Section III-C).

III. Analytes and Labelling Agents

In some embodiments, provided herein are methods and compositions forsample analysis comprising contacting a fixed biological sample that hasbeen catalytically de-crosslinked with a labelling agent that binds toan analyte at a location in the de-crosslinked fixed biological sample,and detecting an optical signal associated with the labelling agent or aproduct thereof, thereby detecting the analyte at the location in thebiological sample. A biological sample may comprise one or a pluralityof analytes of interest. Methods for performing multiplexed assays toanalyze two or more different analytes in a single biological sample areprovided.

The methods, probes, and kits disclosed herein can be used to detect andanalyze a wide variety of different analytes. In some aspects, ananalyte can include any biological substance, structure, moiety, orcomponent to be analyzed. In some aspects, a target disclosed herein maysimilarly include any analyte of interest. In some examples, a target oranalyte can be directly or indirectly detected.

Analytes can be derived from a specific type of cell and/or a specificsub-cellular region. For example, analytes can be derived from cytosol,from cell nuclei, from mitochondria, from microsomes, and moregenerally, from any other compartment, organelle, or portion of a cell.Permeabilizing agents that specifically target certain cell compartmentsand organelles can be used to selectively release analytes from cellsfor analysis, and/or allow access of one or more reagents (e.g., probesfor analyte detection) to the analytes in the cell or cell compartmentor organelle.

The analyte may include any biomolecule, macromolecule, or chemicalcompound, including a protein or peptide, a lipid or a nucleic acidmolecule, or a small molecule, including organic or inorganic molecules.The analyte may be a cell or a microorganism, including a virus, or afragment or product thereof. An analyte can be any substance or entityfor which a specific binding partner (e.g. an affinity binding partner)can be developed. Such a specific binding partner may be a nucleic acidprobe (for a nucleic acid analyte) and may lead directly to thegeneration of a RCA template (e.g. a padlock or other circularizableprobe). Alternatively, the specific binding partner may be coupled to anucleic acid, which may be detected using an RCA strategy, e.g. in anassay which uses or generates a circular nucleic acid molecule which canbe the RCA template.

Analytes of particular interest may include nucleic acid molecules(e.g., cellular nucleic acids), such as DNA (e.g. genomic DNA,mitochondrial DNA, plastid DNA, viral DNA, etc.) and RNA (e.g. mRNA,microRNA, rRNA, snRNA, viral RNA, etc.), and synthetic and/or modifiednucleic acid molecules, (e.g. including nucleic acid domains comprisingor consisting of synthetic or modified nucleotides such as LNA, PNA,morpholino, etc.), proteinaceous molecules such as peptides,polypeptides, proteins or prions or any molecule which comprises aprotein or polypeptide component, etc., or fragments thereof, or a lipidor carbohydrate molecule, or any molecule which comprise a lipid orcarbohydrate component. The analyte may be a single molecule or acomplex that contains two or more molecular subunits, e.g. including butnot limited to protein-DNA complexes, which may or may not be covalentlybound to one another, and which may be the same or different. Thus inaddition to cells or microorganisms, such a complex analyte may also bea protein complex or protein interaction. Such a complex or interactionmay thus be a homo- or hetero-multimer. Aggregates of molecules, e.g.proteins may also be target analytes, for example aggregates of the sameprotein or different proteins. The analyte may also be a complex betweenproteins or peptides and nucleic acid molecules such as DNA or RNA, e.g.interactions between proteins and nucleic acids, e.g. regulatoryfactors, such as transcription factors, and DNA or RNA.

A. Endogenous Analytes

In some embodiments, an analyte herein is endogenous to a biologicalsample and can include cellular nucleic acid analytes and non-nucleicacid analytes. Methods, probes, and kits disclosed herein can be used toanalyze nucleic acid analytes (e.g., using a nucleic acid probe or probeset that directly or indirectly hybridizes to a nucleic acid analyte)and/or non-nucleic acid analytes (e.g., using a labelling agent thatcomprises a reporter oligonucleotide and binds directly or indirectly toa non-nucleic acid analyte) in any suitable combination.

Examples of non-nucleic acid analytes include, but are not limited to,lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked orO-linked), lipoproteins, phosphoproteins, specific phosphorylated oracetylated variants of proteins, amidation variants of proteins,hydroxylation variants of proteins, methylation variants of proteins,ubiquitylation variants of proteins, sulfation variants of proteins,viral coat proteins, extracellular and intracellular proteins,antibodies, and antigen binding fragments. In some embodiments, theanalyte is inside a cell or on a cell surface, such as a transmembraneanalyte or one that is attached to the cell membrane. In someembodiments, the analyte can be an organelle (e.g., nuclei ormitochondria). In some embodiments, the analyte is an extracellularanalyte, such as a secreted analyte. Exemplary analytes include, but arenot limited to, a receptor, an antigen, a surface protein, atransmembrane protein, a cluster of differentiation protein, a proteinchannel, a protein pump, a carrier protein, a phospholipid, aglycoprotein, a glycolipid, a cell-cell interaction protein complex, anantigen-presenting complex, a major histocompatibility complex, anengineered T-cell receptor, a T-cell receptor, a B-cell receptor, achimeric antigen receptor, an extracellular matrix protein, aposttranslational modification (e.g., phosphorylation, glycosylation,ubiquitination, nitrosylation, methylation, acetylation or lipidation)state of a cell surface protein, a gap junction, and an adherensjunction.

Examples of nucleic acid analytes include DNA analytes such assingle-stranded DNA (ssDNA), double-stranded DNA (dsDNA), genomic DNA,methylated DNA, specific methylated DNA sequences, fragmented DNA,mitochondrial DNA, in situ synthesized PCR products, and RNA/DNAhybrids. The DNA analyte can be a transcript of another nucleic acidmolecule (e.g., DNA or RNA such as mRNA) present in a tissue sample.

Examples of nucleic acid analytes also include RNA analytes such asvarious types of coding and non-coding RNA. Examples of the differenttypes of RNA analytes include messenger RNA (mRNA), including a nascentRNA, a pre-mRNA, a primary-transcript RNA, and a processed RNA, such asa capped mRNA (e.g., with a 5′ 7-methyl guanosine cap), a polyadenylatedmRNA (poly-A tail at the 3′ end), and a spliced mRNA in which one ormore introns have been removed. Also included in the analytes disclosedherein are non-capped mRNA, a non-polyadenylated mRNA, and a non-splicedmRNA. The RNA analyte can be a transcript of another nucleic acidmolecule (e.g., DNA or RNA such as viral RNA) present in a tissuesample. Examples of a non-coding RNAs (ncRNA) that is not translatedinto a protein include transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs),as well as small non-coding RNAs such as microRNA (miRNA), smallinterfering RNA (siRNA), Piwi-interacting RNA (piRNA), small nucleolarRNA (snoRNA), small nuclear RNA (snRNA), extracellular RNA (exRNA),small Cajal body-specific RNAs (scaRNAs), and the long ncRNAs such asXist and HOTAIR. The RNA can be small (e.g., less than 200 nucleic acidbases in length) or large (e.g., RNA greater than 200 nucleic acid basesin length). Examples of small RNAs include 5.8S ribosomal RNA (rRNA), 5SrRNA, tRNA, miRNA, siRNA, snoRNAs, piRNA, tRNA-derived small RNA(tsRNA), and small rDNA-derived RNA (srRNA). The RNA can bedouble-stranded RNA or single-stranded RNA. The RNA can be circular RNA.The RNA can be a bacterial rRNA (e.g., 16s rRNA or 23s rRNA).

In some embodiments described herein, an analyte may be a denaturednucleic acid, wherein the resulting denatured nucleic acid issingle-stranded. The nucleic acid may be denatured, for example,optionally using formamide, heat, or both formamide and heat. In someembodiments, the nucleic acid is not denatured for use in a methoddisclosed herein.

Methods, probes, and kits disclosed herein can be used to analyze anynumber of analytes. For example, the number of analytes that areanalyzed can be at least about 2, at least about 3, at least about 4, atleast about 5, at least about 6, at least about 7, at least about 8, atleast about 9, at least about 10, at least about 11, at least about 12,at least about 13, at least about 14, at least about 15, at least about20, at least about 25, at least about 30, at least about 40, at leastabout 50, at least about 100, at least about 1,000, at least about10,000, at least about 100,000 or more different analytes present in aregion of the sample or within an individual feature of the substrate.

In any embodiment described herein, the analyte can comprise or beassociated with a target sequence. In some embodiments, the targetnucleic acid and the target sequence therein may be endogenous to thesample, generated in the sample, added to the sample, or associated withan analyte in the sample. In some embodiments, the target sequence is asingle-stranded target sequence (e.g., a sequence in a rolling circleamplification product). In some embodiments, the target sequence is asingle-stranded target sequence (e.g., in a probe bound directly orindirectly to the analyte). In some embodiments, the target sequence isa single-stranded target sequence in a primary probe that binds to ananalyte of interest in the biological sample. In some embodiments, thetarget sequence is a single-stranded target sequence in an intermediateprobe which directly or indirectly binds to a primary probe or productthereof, where the primary probe binds to an analyte of interest in thebiological sample. In some embodiments, the target sequence is asingle-stranded target sequence in a secondary probe that binds to theprimary probe or product thereof. In some embodiments, the analytescomprises one or more single-stranded target sequences.

B. Analyte Detection

In some embodiments, provided herein are methods, probes, and kits foranalyzing one or more products of an endogenous analyte and/or alabelling agent in a biological sample. In some embodiments, anendogenous analyte (e.g., a viral or cellular DNA or RNA) or a product(e.g., a hybridization product, a ligation product, an extension product(e.g., by a DNA or RNA polymerase), a replication product, atranscription/reverse transcription product, and/or an amplificationproduct such as a rolling circle amplification (RCA) product) thereof isanalyzed. In some embodiments, a labelling agent that directly orindirectly binds to an analyte in the biological sample is analyzed. Insome embodiments, a product (e.g., a hybridization product, a ligationproduct, an extension product (e.g., by a DNA or RNA polymerase), areplication product, a transcription/reverse transcription product,and/or an amplification product such as a rolling circle amplification(RCA) product) of a labelling agent that directly or indirectly binds toan analyte in the biological sample is analyzed. In some instances, theprovided methods are for gene expression analysis for RNA transcriptsand protein analysis in the same intact biological sample (e.g., atissue section).

Disclosed herein in some aspects are labelling agents (e.g., nucleicacid probes and/or probe sets) that are introduced into a cell or usedto otherwise contact a biological sample such as a tissue sample. Thelabelling agents include probes (e.g., the primary probes disclosedherein and/or any detectable probe disclosed herein) may comprise any ofa variety of entities that can hybridize to a nucleic acid, typically byWatson-Crick base pairing, such as DNA, RNA, LNA, PNA, etc. The nucleicacid probe may comprise a hybridization region that is able to directlyor indirectly bind to at least a portion of a target sequence in atarget nucleic acid. The nucleic acid probe may be able to bind to aspecific target nucleic acid (e.g., an mRNA, or other nucleic acidsdisclosed herein). In some embodiments, the nucleic acid probes may bedetected using a detectable label, and/or by using secondary nucleicacid probes able to bind to the nucleic acid probes. In someembodiments, the nucleic acid probes (e.g., primary probes and/orsecondary probes) are compatible with one or more biological and/orchemical reactions. For instance, a nucleic acid probe disclosed hereincan serve as a template or primer for a polymerase, a template orsubstrate for a ligase, a substrate for a click chemistry reaction,and/or a substrate for a nuclease (e.g., endonuclease or exonuclease forcleavage or digestion).

In some embodiments, more than one type of primary nucleic acid probesmay be contacted with a sample, e.g., simultaneously or sequentially inany suitable order, such as in sequential probehybridization/unhybridization cycles. In some embodiments, more than onetype of secondary nucleic acid probes may be contacted with a sample,e.g., simultaneously or sequentially in any suitable order, such as insequential probe hybridization/unhybridization cycles. In someembodiments, the secondary probes may comprise probes that bind to aproduct of a primary probe targeting an analyte. In some embodiments,more than one type of higher order nucleic acid probes may be contactedwith a sample, e.g., simultaneously or sequentially in any suitableorder, such as in sequential probe hybridization/unhybridization cycles.In some embodiments, more than one type of detectably labeled nucleicacid probes may be contacted with a sample, e.g., simultaneously orsequentially in any suitable order, such as in sequential probehybridization/unhybridization cycles. In some embodiments, thedetectably labeled nucleic acid probes can be used to bind to one ormore primary probes, one or more secondary probes, one or more higherorder probes, one or more intermediate probes between aprimary/secondary/higher order probes, and/or one or more detectably ornon-detectably labeled probes (e.g., as in the case of a hybridizationchain reaction (HCR), a branched DNA reaction (bDNA), or the like). Insome embodiments, the plurality of probes or probe sets comprises atleast 2, at least 5, at least 10, at least 25, at least 50, at least 75,at least 100, at least 300, at least 1,000, at least 3,000, at least10,000, at least 30,000, at least 50,000, at least 100,000, at least250,000, at least 500,000, or at least 1,000,000 distinguishable nucleicacid probes (e.g., primary, secondary, higher order probes, and/ordetectably labeled probes) that can be contacted with a sample, e.g.,simultaneously or sequentially in any suitable order. Between any of theprobe contacting steps disclosed herein, the method may comprise one ormore intervening reactions and/or processing steps, such asmodifications of a target nucleic acid, modifications of a probe orproduct thereof (e.g., via hybridization, ligation, extension,amplification, cleavage, digestion, branch migration, primer exchangereaction, click chemistry reaction, crosslinking, attachment of adetectable label, activating photo-reactive moieties, etc.), removal ofa probe or product thereof (e.g., cleaving off a portion of a probeand/or unhybridizing the entire probe), signal modifications (e.g.,quenching, masking, photo-bleaching, signal enhancement (e.g., viaFRET), signal amplification, etc.), signal removal (e.g., cleaving offor permanently inactivating a detectable label), crosslinking,de-crosslinking, and/or signal detection.

The hybridization region of the probe or probe set is a target-bindingsequence (sometimes also referred to as the targeting region/sequence orthe recognition region/sequence) that be positioned anywhere within theprobe. For instance, the target-binding sequence of a primary probe thatbinds to a target nucleic acid can be 5′ or 3′ to any barcode sequencein the primary probe. Likewise, the target-binding sequence of asecondary probe (which binds to a primary probe or complement or productthereof) can be 5′ or 3′ to any barcode sequence in the secondary probe.In some embodiments, the target-binding sequence may comprise a sequencethat is substantially complementary to a portion of a target nucleicacid. In some embodiments, the portions may be at least 50%, at least60%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 92%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% complementary.

The hybridization region of the probe or probe set can be used toidentify a particular analyte comprising or associated with a target(e.g., comprising a target sequence). In some cases, multiple probes canbe used, sequentially and/or simultaneously, that can bind to (e.g.,hybridize to) different regions of the same target nucleic acid. Inother examples, a probe may comprise target-binding sequences (e.g.,hybridization regions) that can bind to different target nucleic acidsequences, e.g., various intron and/or exon sequences of the same gene(for detecting splice variants, for example), or sequences of differentgenes, e.g., for detecting a product that comprises the different targetnucleic acid sequences, such as a genome rearrangement (e.g., inversion,transposition, translocation, insertion, deletion, duplication, and/oramplification).

In some embodiments, provided herein are methods, probes, and kits foranalyzing endogenous analytes (e.g., RNA, ssDNA, and cell surface orintracellular proteins and/or metabolites) in a sample using one or morelabelling agents.

In some embodiments, the labelling agent is an immunohistochemistry(IHC) probe that is excited at various different wavelengths. In someembodiments, an analyte labelling agent may include an agent thatinteracts with an analyte (e.g., an endogenous analyte in a sample). Insome embodiments, the labelling agents can comprise a reporteroligonucleotide that is indicative of the analyte or portion thereofinteracting with the labelling agent. For example, the reporteroligonucleotide may comprise a barcode sequence that permitsidentification of the labelling agent. In some cases, the samplecontacted by the labelling agent can be further contacted with a probe(e.g., a single-stranded probe sequence), that hybridizes to a reporteroligonucleotide of the labelling agent, in order to identify the analyteassociated with the labelling agent. In some embodiments, the analytelabelling agent comprises an analyte binding moiety and a labellingagent barcode domain comprising one or more barcode sequences, e.g., abarcode sequence that corresponds to the analyte binding moiety and/orthe analyte. An analyte binding moiety barcode comprises to a barcodethat is associated with or otherwise identifies the analyte bindingmoiety. In some embodiments, by identifying an analyte binding moiety byidentifying its associated analyte binding moiety barcode, the analyteto which the analyte binding moiety binds can also be identified. Ananalyte binding moiety barcode can be a nucleic acid sequence of a givenlength and/or sequence that is associated with the analyte bindingmoiety. An analyte binding moiety barcode can generally include any ofthe variety of aspects of barcodes described herein.

In some embodiments, the method comprises one or more post-fixing (alsoreferred to as post-fixation) steps after contacting the sample with oneor more labelling agents.

In the methods described herein, one or more labelling agents capable ofbinding to or otherwise coupling to one or more features may be used tocharacterize analytes, cells and/or cell features. In some instances,cell features include cell surface features. Analytes may include, butare not limited to, a protein, a receptor, an antigen, a surfaceprotein, a transmembrane protein, a cluster of differentiation protein,a protein channel, a protein pump, a carrier protein, a phospholipid, aglycoprotein, a glycolipid, a cell-cell interaction protein complex, anantigen-presenting complex, a major histocompatibility complex, anengineered T-cell receptor, a T-cell receptor, a B-cell receptor, achimeric antigen receptor, a gap junction, an adherens junction, or anycombination thereof. In some instances, cell features may includeintracellular analytes, such as proteins, protein modifications (e.g.,phosphorylation status or other post-translational modifications),nuclear proteins, nuclear membrane proteins, or any combination thereof.

In some embodiments, an analyte binding moiety may include any moleculeor moiety capable of binding to an analyte (e.g., a biological analyte,e.g., a macromolecular constituent). A labelling agent may include, butis not limited to, a protein, a peptide, an antibody (or an epitopebinding fragment thereof), a lipophilic moiety (such as cholesterol), acell surface receptor binding molecule, a receptor ligand, a smallmolecule, a bi-specific antibody, a bi-specific T-cell engager, a T-cellreceptor engager, a B-cell receptor engager, a pro-body, an aptamer, amonobody, an affimer, a darpin, and a protein scaffold, or anycombination thereof. The labelling agents can include (e.g., areattached to) a reporter oligonucleotide that is indicative of the cellsurface feature to which the binding group binds. For example, thereporter oligonucleotide may comprise a barcode sequence that permitsidentification of the labelling agent. For example, a labelling agentthat is specific to one type of cell feature (e.g., a first cell surfacefeature) may have coupled thereto a first reporter oligonucleotide,while a labelling agent that is specific to a different cell feature(e.g., a second cell surface feature) may have a different reporteroligonucleotide coupled thereto. For a description of exemplarylabelling agents, reporter oligonucleotides, and methods of use, see,e.g., U.S. Pat. No. 10,550,429; U.S. Pat. Pub. 20190177800; and U.S.Pat. Pub. 20190367969, which are each incorporated by reference hereinin their entirety.

In some embodiments, an analyte binding moiety comprises one or moreantibodies or antigen binding fragments thereof. The antibodies orantigen binding fragments including the analyte binding moiety canspecifically bind to a target analyte. In some embodiments, the analyteis a protein (e.g., a protein on a surface of the biological sample(e.g., a cell) or an intracellular protein). In some embodiments, aplurality of analyte labelling agents comprising a plurality of analytebinding moieties bind a plurality of analytes present in a biologicalsample. In some embodiments, the plurality of analytes comprises asingle species of analyte (e.g., a single species of polypeptide). Insome embodiments in which the plurality of analytes comprises a singlespecies of analyte, the analyte binding moieties of the plurality ofanalyte labelling agents are the same. In some embodiments in which theplurality of analytes comprises a single species of analyte, the analytebinding moieties of the plurality of analyte labelling agents are thedifferent (e.g., members of the plurality of analyte labelling agentscan have two or more species of analyte binding moieties, wherein eachof the two or more species of analyte binding moieties binds a singlespecies of analyte, e.g., at different binding sites). In someembodiments, the plurality of analytes comprises multiple differentspecies of analyte (e.g., multiple different species of polypeptides).

In other instances, e.g., to facilitate sample multiplexing, a labellingagent that is specific to a particular cell feature may have a firstplurality of the labelling agent (e.g., an antibody or lipophilicmoiety) coupled to a first reporter oligonucleotide and a secondplurality of the labelling agent coupled to a second reporteroligonucleotide.

In some aspects, these reporter oligonucleotides may comprise nucleicacid barcode sequences that permit identification of the labelling agentwhich the reporter oligonucleotide is coupled to. The selection ofoligonucleotides as the reporter may provide advantages of being able togenerate significant diversity in terms of sequence, while also beingreadily attachable to most biomolecules, e.g., antibodies, etc., as wellas being readily detected.

Attachment (coupling) of the reporter oligonucleotides to the labellingagents may be achieved through any of a variety of direct or indirect,covalent or non-covalent associations or attachments. For example,oligonucleotides may be covalently attached to a portion of a labellingagent (such a protein, e.g., an antibody or antibody fragment) usingchemical conjugation techniques (e.g., Lightning-Link® antibodylabelling kits available from Innova Biosciences), as well as othernon-covalent attachment mechanisms, e.g., using biotinylated antibodiesand oligonucleotides (or beads that include one or more biotinylatedlinker, coupled to oligonucleotides) with an avidin or streptavidinlinker. Antibody and oligonucleotide biotinylation techniques areavailable. See, e.g., Fang, et al., “Fluoride-Cleavable BiotinylationPhosphoramidite for 5′-end-Labelling and Affinity Purification ofSynthetic Oligonucleotides,” Nucleic Acids Res. Jan. 15, 2003;31(2):708-715, which is entirely incorporated herein by reference forall purposes. Likewise, protein and peptide biotinylation techniqueshave been developed and are readily available. See, e.g., U.S. Pat. No.6,265,552, which is entirely incorporated herein by reference for allpurposes. Furthermore, click reaction chemistry may be used to couplereporter oligonucleotides to labelling agents. Commercially availablekits, such as those from Thunderlink and Abcam, and techniques common inthe art may be used to couple reporter oligonucleotides to labellingagents as appropriate. In another example, a labelling agent isindirectly (e.g., via hybridization) coupled to a reporteroligonucleotide comprising a barcode sequence that identifies the labelagent. For instance, the labelling agent may be directly coupled (e.g.,covalently bound) to a hybridization oligonucleotide that comprises asequence that hybridizes with a sequence of the reporteroligonucleotide. Hybridization of the hybridization oligonucleotide tothe reporter oligonucleotide couples the labelling agent to the reporteroligonucleotide. In some embodiments, the reporter oligonucleotides arereleasable from the labelling agent, such as upon application of astimulus. For example, the reporter oligonucleotide may be attached tothe labelling agent through a labile bond (e.g., chemically labile,photolabile, thermally labile, etc.) as generally described forreleasing molecules from supports elsewhere herein.

In some cases, the labelling agent can comprise a reporteroligonucleotide and a label. A label can be fluorophore, a radioisotope,a molecule capable of a colorimetric reaction, a magnetic particle, orany other suitable molecule or compound capable of detection. The labelcan be conjugated to a labelling agent (or reporter oligonucleotide)either directly or indirectly (e.g., the label can be conjugated to amolecule that can bind to the labelling agent or reporteroligonucleotide). In some cases, a label is conjugated to a firstoligonucleotide that is complementary (e.g., hybridizes) to a sequenceof the reporter oligonucleotide.

In some embodiments, multiple different species of analytes (e.g.,polypeptides) from the biological sample can be subsequently associatedwith the one or more physical properties of the biological sample. Forexample, the multiple different species of analytes can be associatedwith locations of the analytes in the biological sample. Suchinformation (e.g., proteomic information when the analyte bindingmoiety(ies) recognizes a polypeptide(s)) can be used in association withother spatial information (e.g., genetic information from the biologicalsample, such as DNA sequence information, transcriptome information(e.g., sequences of transcripts), or both). For example, a cell surfaceprotein of a cell can be associated with one or more physical propertiesof the cell (e.g., a shape, size, activity, or a type of the cell). Theone or more physical properties can be characterized by imaging thecell. The cell can be bound by an analyte labelling agent comprising ananalyte binding moiety that binds to the cell surface protein and ananalyte binding moiety barcode that identifies that analyte bindingmoiety. Results of protein analysis in a sample (e.g., a tissue sampleor a cell) can be associated with DNA and/or RNA analysis in the sample.

a. Hybridization

In some embodiments, the labelling agents (e.g., probes or probes sets)described herein can be used to detect an endogenous analyte, a productof an endogenous analyte and/or a labelling agent is a hybridizationproduct comprising the pairing of substantially complementary orcomplementary nucleic acid sequences within two different molecules, oneof which is the endogenous analyte or the labelling agent (e.g.,reporter oligonucleotide attached thereto). The other molecule can beanother endogenous molecule or an exogenous molecule such as a probe.Pairing can be achieved by any process in which a nucleic acid sequencejoins with a substantially or fully complementary sequence through basepairing to form a hybridization complex. For purposes of hybridization,two nucleic acid sequences are “substantially complementary” if at least60% (e.g., at least 70%, at least 80%, or at least 90%) of theirindividual bases are complementary to one another.

Various probes and probe sets can be hybridized to an endogenous analyteand/or a labelling agent and each probe may comprise one or more barcodesequences. In some instances, various probes and probe sets can be usedto generate a product comprising a target sequence that can behybridized by one or more detectable probes. In some instances, a probeor probe set disclosed herein is a circularizable probe or probe setcomprising a barcode region comprising one or more barcode sequences.Exemplary barcoded probes or probe sets may be based on a padlock probe,a gapped padlock probe, a SNAIL (Splint Nucleotide AssistedIntramolecular Ligation) probe set, a PLAYR (Proximity Ligation Assayfor RNA) probe set, a PLISH (Proximity Ligation in situ Hybridization)probe set, and RNA-templated ligation probes. The specific probe orprobe set design can vary.

b. Ligation

In some embodiments, a product of an endogenous analyte and/or alabelling agent is a ligation product that may comprise a targetsequence that can be hybridized by one or more probes described herein.In some embodiments, the ligation product is formed between two or moreendogenous analytes. In some embodiments, the ligation product is formedbetween an endogenous analyte and a labelling agent. In someembodiments, the ligation product is formed between two or morelabelling agent. In some embodiments, the ligation product is anintramolecular ligation of an endogenous analyte. In some embodiments,the ligation product is an intramolecular ligation of a labelling agentor probe, for example, the circularization of a circularizable probe orprobe set upon hybridization to a target sequence. The target sequencecan be comprised in an endogenous analyte (e.g., nucleic acid such asgenomic DNA or mRNA) or a product thereof (e.g., cDNA from a cellularmRNA transcript), or in a labelling agent (e.g., the reporteroligonucleotide) or a product thereof.

In some embodiments, provided herein is a labelling agent comprising aprobe or probe set capable of DNA-templated ligation, such as from acDNA molecule. See, e.g., U.S. Pat. No. 8,551,710, which is herebyincorporated by reference in its entirety. In some embodiments, providedherein is a probe or probe set capable of RNA-templated ligation. See,e.g., U.S. Pat. Pub. 2020/0224244 which is hereby incorporated byreference in its entirety. In some embodiments, the probe set is a SNAILprobe set. See, e.g., U.S. Pat. Pub. 20190055594, which is herebyincorporated by reference in its entirety.

In some embodiments, provided herein is a multiplexed proximity ligationassay. See, e.g., U.S. Pat. Pub. 20140194311 which is herebyincorporated by reference in its entirety. In some embodiments, providedherein is a probe or probe set capable of proximity ligation, forinstance a proximity ligation assay for RNA (e.g., PLAYR) probe set.See, e.g., U.S. Pat. Pub. 20160108458, which is hereby incorporated byreference in its entirety. In some embodiments, a circular probe can beindirectly hybridized to the target nucleic acid. In some embodiments,the circular construct is formed from a probe set capable of proximityligation, for instance a proximity ligation in situ hybridization(PLISH) probe set. See, e.g., U.S. Pat. Pub. 2020/0224243 which ishereby incorporated by reference in its entirety.

In some embodiments, a circular or circularizable probe or probe set maybe used to analyze a reporter oligonucleotide, which may generated usingproximity ligation or be subjected to proximity ligation. In someexamples, the reporter oligonucleotide of a labelling agent thatspecifically recognizes a protein can be analyzed using in situhybridization (e.g., sequential hybridization) and/or in situ sequencing(e.g., using circular or circularizable probes and rolling circleamplification of circular or circularized probes). Further, the reporteroligonucleotide of the labelling agent and/or a complement thereofand/or a product (e.g., a hybridization product, a ligation product, anextension product (e.g., by a DNA or RNA polymerase), a replicationproduct, a transcription/reverse transcription product, and/or anamplification product) thereof can be recognized by another labellingagent and analyzed.

In some embodiments, an analyte (a nucleic acid analyte or non-nucleicacid analyte) can be specifically bound by two labelling agents (e.g.,antibodies) each of which is attached to a reporter oligonucleotide(e.g., DNA) that can participate in ligation, replication, and sequencedecoding reactions, e.g., using a probe or probe set (e.g., a padlockprobe, a SNAIL probe set, a circular probe, a gapped padlock probe, or agapped padlock probe and a connector). In some embodiments, the probeset may comprise two or more probe oligonucleotides, each comprising aregion that is complementary to each other. For example, a proximityligation reaction can include reporter oligonucleotides attached topairs of antibodies that can be joined by ligation if the antibodieshave been brought in proximity to each other, e.g., by binding the sametarget protein (complex), and the DNA ligation products that form arethen used to template PCR amplification, as described for example inSöderberg et al., Methods. (2008), 45(3): 227-32, the entire contents ofwhich are incorporated herein by reference. In some embodiments, aproximity ligation reaction can include reporter oligonucleotidesattached to antibodies that each bind to one member of a binding pair orcomplex, for example, for analyzing a binding between members of thebinding pair or complex. For detection of analytes usingoligonucleotides in proximity, see, e.g., U.S. Patent ApplicationPublication No. 2002/0051986, the entire contents of which areincorporated herein by reference. In some embodiments, two analytes inproximity can be specifically bound by two labelling agents (e.g.,antibodies) each of which is attached to a reporter oligonucleotide(e.g., DNA) that can participate, when in proximity when bound to theirrespective targets, in ligation, replication, and/or sequence decodingreactions.

In some embodiments, one or more reporter oligonucleotides (andoptionally one or more other nucleic acid molecules such as a connector)aid in the ligation of the probe. Upon ligation, the probe may form acircularized probe. In some embodiments, one or more suitable probes canbe used and ligated, wherein the one or more probes comprise a sequencethat is complementary to the one or more reporter oligonucleotides (orportion thereof). The probe may comprise one or more barcode sequences.In some embodiments, the one or more reporter oligonucleotide may serveas a primer for rolling circle amplification (RCA) of the circularizedprobe. In some embodiments, a nucleic acid other than the one or morereporter oligonucleotide is used as a primer for rolling circleamplification (RCA) of the circularized probe. For example, a nucleicacid capable of hybridizing to the circularized probe at a sequenceother than sequence(s) hybridizing to the one or more reporteroligonucleotide can be used as the primer for RCA. In other examples,the primer in a SNAIL probe set is used as the primer for RCA.

In some embodiments, one or more analytes can be specifically bound bytwo primary antibodies, each of which is in turn recognized by asecondary antibody each attached to a reporter oligonucleotide (e.g.,DNA). Each nucleic acid molecule can aid in the ligation of the probe toform a circularized probe. In some instances, the probe can comprise oneor more barcode sequences. Further, the reporter oligonucleotide mayserve as a primer for rolling circle amplification of the circularizedprobe. The nucleic acid molecules, circularized probes, and RCA productscan be analyzed using any suitable method disclosed herein for in situanalysis.

In some embodiments, the ligation involves chemical ligation. In someembodiments, the ligation involves template dependent ligation. In someembodiments, the ligation involves template independent ligation. Insome embodiments, the ligation involves enzymatic ligation.

In some embodiments, the enzymatic ligation involves use of a ligase. Insome aspects, the ligase used herein comprises an enzyme that iscommonly used to join polynucleotides together or to join the ends of asingle polynucleotide. An RNA ligase, a DNA ligase, or another varietyof ligase can be used to ligate two nucleotide sequences together.Ligases comprise ATP-dependent double-strand polynucleotide ligases,NAD-i-dependent double-strand DNA or RNA ligases and single-strandpolynucleotide ligases, for example any of the ligases described in EC6.5.1.1 (ATP-dependent ligases), EC 6.5.1.2 (NAD+-dependent ligases), EC6.5.1.3 (RNA ligases). Specific examples of ligases comprise bacterialligases such as E. coli DNA ligase, Tth DNA ligase, Thermococcus sp.(strain 9° N) DNA ligase (9° N™ DNA ligase, New England Biolabs), TaqDNA ligase, Ampligase™ (Epicentre Biotechnologies) and phage ligasessuch as T3 DNA ligase, T4 DNA ligase and T7 DNA ligase and mutantsthereof. In some embodiments, the ligase is a T4 RNA ligase. In someembodiments, the ligase is a splintR ligase. In some embodiments, theligase is a single stranded DNA ligase. In some embodiments, the ligaseis a T4 DNA ligase. In some embodiments, the ligase is a ligase that hasan DNA-splinted DNA ligase activity. In some embodiments, the ligase isa ligase that has an RNA-splinted DNA ligase activity.

In some embodiments, the ligation herein is a direct ligation. In someembodiments, the ligation herein is an indirect ligation. “Directligation” means that the ends of the polynucleotides hybridizeimmediately adjacently to one another to form a substrate for a ligaseenzyme resulting in their ligation to each other (intramolecularligation). Alternatively, “indirect” means that the ends of thepolynucleotides hybridize non-adjacently to one another, e.g., separatedby one or more intervening nucleotides or “gaps”. In some embodiments,said ends are not ligated directly to each other, but instead occurseither via the intermediacy of one or more intervening (so-called “gap”or “gap-filling” (oligo)nucleotides) or by the extension of the 3′ endof a probe to “fill” the “gap” corresponding to said interveningnucleotides (intermolecular ligation). In some cases, the gap of one ormore nucleotides between the hybridized ends of the polynucleotides maybe “filled” by one or more “gap” (oligo)nucleotide(s) which arecomplementary to a splint, padlock probe, or target nucleic acid. Thegap may be a gap of 1 to 60 nucleotides or a gap of 1 to 40 nucleotidesor a gap of 3 to 40 nucleotides. In specific embodiments, the gap may bea gap of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides, ofany integer (or range of integers) of nucleotides in between theindicated values. In some embodiments, the gap between said terminalregions may be filled by a gap oligonucleotide or by extending the 3′end of a polynucleotide. In some cases, ligation involves ligating theends of the probe to at least one gap (oligo)nucleotide, such that thegap (oligo)nucleotide becomes incorporated into the resultingpolynucleotide. In some embodiments, the ligation herein is preceded bygap filling. In other embodiments, the ligation herein does not requiregap filling.

In some embodiments, ligation of the polynucleotides producespolynucleotides with melting temperature higher than that of unligatedpolynucleotides. Thus, in some aspects, ligation stabilizes thehybridization complex containing the ligated polynucleotides prior tosubsequent steps, comprising amplification and detection.

In some aspects, a high fidelity ligase, such as a thermostable DNAligase (e.g., a Taq DNA ligase), is used. Thermostable DNA ligases areactive at elevated temperatures, allowing further discrimination byincubating the ligation at a temperature near the melting temperature(T_(m)) of the DNA strands. This selectively reduces the concentrationof annealed mismatched substrates (expected to have a slightly lowerT_(m) around the mismatch) over annealed fully base-paired substrates.Thus, high-fidelity ligation can be achieved through a combination ofthe intrinsic selectivity of the ligase active site and balancedconditions to reduce the incidence of annealed mismatched dsDNA.

In some embodiments, the ligation herein is a proximity ligation ofligating two (or more) nucleic acid sequences that are in proximity witheach other, e.g., through enzymatic means (e.g., a ligase). In someembodiments, proximity ligation can include a “gap-filling” step thatinvolves incorporation of one or more nucleic acids by a polymerase,based on the nucleic acid sequence of a template nucleic acid molecule,spanning a distance between the two nucleic acid molecules of interest(see, e.g., U.S. Pat. No. 7,264,929, the entire contents of which areincorporated herein by reference). A wide variety of different methodscan be used for proximity ligating nucleic acid molecules, including(but not limited to) “sticky-end” and “blunt-end” ligations.Additionally, single-stranded ligation can be used to perform proximityligation on a single-stranded nucleic acid molecule. Sticky-endproximity ligations involve the hybridization of complementarysingle-stranded sequences between the two nucleic acid molecules to bejoined, prior to the ligation event itself. Blunt-end proximityligations generally do not include hybridization of complementaryregions from each nucleic acid molecule because both nucleic acidmolecules lack a single-stranded overhang at the site of ligation.

c. Primer Extension

In some embodiments, a primer extension product of an analyte, alabelling agent, a probe or probe set bound to the analyte (e.g., boundto genomic DNA, mRNA, or cDNA), or a probe or probe set bound to thelabelling agent (e.g., bound to one or more reporter oligonucleotidesfrom the same or different labelling agents). Any of such products ofextension may comprise a target sequence that can be hybridized by theplurality of probes or probe sets described herein.

In some embodiments, the plurality of probes or probe sets comprises aprimer. A primer is generally a single-stranded nucleic acid sequencehaving a 3′ end that can be used as a substrate for a nucleic acidpolymerase in a nucleic acid extension reaction. RNA primers are formedof RNA nucleotides, and are used in RNA synthesis, while DNA primers areformed of DNA nucleotides and used in DNA synthesis. Primers can alsoinclude both RNA nucleotides and DNA nucleotides (e.g., in a random ordesigned pattern). Primers can also include other natural or syntheticnucleotides described herein that can have additional functionality. Insome examples, DNA primers can be used to prime RNA synthesis and viceversa (e.g., RNA primers can be used to prime DNA synthesis). Primerscan vary in length. For example, primers can be about 6 bases to about120 bases. For example, primers can include up to about 25 bases. Aprimer, may in some cases, refer to a primer binding sequence. A primerextension reaction generally refers to any method where two nucleic acidsequences become linked (e.g., hybridized) by an overlap of theirrespective terminal complementary nucleic acid sequences (e.g., forexample, 3′ termini). Such linking can be followed by nucleic acidextension (e.g., an enzymatic extension) of one, or both termini usingthe other nucleic acid sequence as a template for extension. Enzymaticextension can be performed by an enzyme including, but not limited to, apolymerase and/or a reverse transcriptase.

In some embodiments, a product of an endogenous analyte and/or alabelling agent is an amplification product of one or morepolynucleotides, for instance, a circular probe or circularizable probeor probe set. In some embodiments, the amplifying is achieved byperforming rolling circle amplification (RCA). In other embodiments, aprimer that hybridizes to the circular probe or circularized probe isadded and used as such for amplification. In some embodiments, the RCAcomprises a linear RCA, a branched RCA, a dendritic RCA, or anycombination thereof.

In some embodiments, the amplification is performed at a temperaturebetween or between about 20° C. and about 60° C. In some embodiments,the amplification is performed at a temperature between or between about30° C. and about 40° C. In some aspects, the amplification step, such asthe rolling circle amplification (RCA) is performed at a temperaturebetween at or about 25° C. and at or about 50° C., such as at or about25° C., 27° C., 29° C., 31° C., 33° C., 35° C., 37° C., 39° C., 41° C.,43° C., 45° C., 47° C., or 49° C.

In some embodiments, upon addition of a DNA polymerase in the presenceof appropriate dNTP precursors and other cofactors, a primer iselongated to produce multiple copies of the circular template. Thisamplification step can utilize isothermal amplification ornon-isothermal amplification. In some embodiments, after the formationof the hybridization complex and association of the amplification probe,the hybridization complex is rolling-circle amplified to generate a cDNAnanoball (e.g., amplicon) containing multiple copies of the cDNA.Techniques for rolling circle amplification (RCA) include linear RCA, abranched RCA, a dendritic RCA, or any combination thereof. See, e.g.,Baner et a1, Nucleic Acids Research, 26:5073-5078, 1998; Lizardi et a1,Nature Genetics 19:226, 1998; Mohsen et al., Acc Chem Res. 2016 Nov. 15;49(11): 2540-2550; Schweitzer et a1. Proc. Natl Acad. Sci. USA97(18):10113-9, 2000; Faruqi et a1, BMC Genomics 2:4, 2000; Nallur eta1, Nucl. Acids Res. 29:e118, 2001; Dean et a1. Genome Res.11:1095-1099, 2001; Schweitzer et a1, Nature Biotech. 20:359-365, 2002;U.S. Pat. Nos. 6,054,274, 6,291,187, 6,323,009, 6,344,329 and 6,368,801,all of which are incorporated by reference. Exemplary polymerases foruse in RCA comprise DNA polymerase such phi29 (φ29) polymerase, Klenowfragment, Bacillus stearothermophilus DNA polymerase (BST), T4 DNApolymerase, T7 DNA polymerase, or DNA polymerase I. In some aspects, DNApolymerases that have been engineered or mutated to have desirablecharacteristics can be employed. In some embodiments, the polymerase isphi29 DNA polymerase.

In some aspects, during the amplification step, modified nucleotides canbe added to the reaction to incorporate the modified nucleotides in theamplification product (e.g., nanoball). Exemplary of the modifiednucleotides comprise amine-modified nucleotides. In some aspects of themethods, for example, for anchoring or cross-linking of the generatedamplification product (e.g., nanoball) to a scaffold, to cellularstructures and/or to other amplification products (e.g., othernanoballs). In some aspects, the amplification products comprises amodified nucleotide, such as an amine-modified nucleotide. In someembodiments, the amine-modified nucleotide comprises an acrylic acidN-hydroxysuccinimide moiety modification. Examples of otheramine-modified nucleotides comprise, but are not limited to, a5-Aminoallyl-dUTP moiety modification, a 5-Propargylamino-dCTP moietymodification, a N⁶-6-Aminohexyl-dATP moiety modification, or a7-Deaza-7-Propargylamino-dATP moiety modification.

In some aspects, the polynucleotides and/or amplification product (e.g.,amplicon) can be anchored to a polymer matrix. For example, the polymermatrix can be a hydrogel. In some embodiments, one or more of thepolynucleotide probe(s) can be modified to contain functional groupsthat can be used as an anchoring site to attach the polynucleotideprobes and/or amplification product to a polymer matrix. Exemplarymodification and polymer matrix that can be employed in accordance withthe provided embodiments comprise those described in, for example, US2016/0024555, US 2018/0251833, US 2017/0219465, U.S. Pat. Nos.10,138,509, 10,494,662, 11,078,520, 11,299,767, 10,266,888, 11,118,220,US 2021/0363579, US 2021/0324450, and US 2021/0215581, all of which areherein incorporated by reference in their entireties. In some examples,the scaffold also contains modifications or functional groups that canreact with or incorporate the modifications or functional groups of theprobe set or amplification product. In some examples, the scaffold cancomprise oligonucleotides, polymers or chemical groups, to provide amatrix and/or support structures.

The amplification products may be immobilized within the matrixgenerally at the location of the nucleic acid being amplified, therebycreating a localized colony of amplicons. The amplification products maybe immobilized within the matrix by steric factors. The amplificationproducts may also be immobilized within the matrix by covalent ornoncovalent bonding. In this manner, the amplification products may beconsidered to be attached to the matrix. By being immobilized to thematrix, such as by covalent bonding or cross-linking, the size andspatial relationship of the original amplicons is maintained. By beingimmobilized to the matrix, such as by covalent bonding or cross-linking,the amplification products are resistant to movement or unraveling undermechanical stress.

In some aspects, the amplification products are copolymerized and/orcovalently attached to the surrounding matrix thereby preserving theirspatial relationship and any information inherent thereto. For example,if the amplification products are those generated from DNA or RNA withina cell embedded in the matrix, the amplification products can also befunctionalized to form covalent attachment to the matrix preservingtheir spatial information within the cell thereby providing asubcellular localization distribution pattern. In some embodiments, theprovided methods involve embedding the one or more polynucleotide probesets and/or the amplification products in the presence of hydrogelsubunits to form one or more hydrogel-embedded amplification products.In some embodiments, the hydrogel-tissue chemistry described comprisescovalently attaching nucleic acids to in situ synthesized hydrogel fortissue clearing, enzyme diffusion, and multiple-cycle sequencing whilean existing hydrogel-tissue chemistry method cannot. In someembodiments, to enable amplification product embedding in thetissue-hydrogel setting, amine-modified nucleotides are comprised in theamplification step (e.g., RCA), functionalized with an acrylamide moietyusing acrylic acid N-hydroxysuccinimide esters, and copolymerized withacrylamide monomers to form a hydrogel.

In some embodiments, the RCA template may comprise the target analyte,or a part thereof, where the target analyte is a nucleic acid, or it maybe provided or generated as a proxy, or a marker, for the analyte. Asnoted above, the detection of numerous different analytes may use aRCA-based detection system, e.g., where the signal is provided bygenerating a target sequence from a circular RCA template which isprovided or generated in the assay, and the target sequence is detectedto detect the corresponding analyte. The target sequence may thus beregarded as a reporter which is detected to detect the target analyte.However, the RCA template may also be regarded as a reporter for thetarget analyte; the target sequence is generated based on the RCAtemplate, and comprises complementary copies of the RCA template. TheRCA template determines the signal which is detected, and is thusindicative of the target analyte. As will be described in more detailbelow, the RCA template may be a probe, or a part or component of aprobe, or may be generated from a probe, or it may be a component of adetection assay (e.g. a reagent in a detection assay), which is used asa reporter for the assay, or a part of a reporter, or signal-generationsystem. The RCA template used to generate the target sequence may thusbe a circular (e.g. circularized) reporter nucleic acid molecule, namelyfrom any RCA-based detection assay which uses or generates a circularnucleic acid molecule as a reporter for the assay. Since the RCAtemplate generates the target sequence reporter, it may be viewed aspart of the reporter system for the assay.

In some embodiments, a product herein comprises a molecule or a complexgenerated in a series of reactions, e.g., hybridization, ligation,extension, replication, transcription/reverse transcription, and/oramplification (e.g., rolling circle amplification), in any suitablecombination. For example, a product comprising a target sequence for atarget-binding region in a probe may be a hybridization complex formedof a cellular nucleic acid in a sample and an exogenously added nucleicacid probe or a product generated therefrom. The exogenously addednucleic acid probe (e.g., plurality of probes or probe sets) maycomprise an overhang that does not hybridize to the cellular nucleicacid but hybridizes to another probe (e.g., an intermediate probe).

In some embodiments, the labelling agents may bind or hybridize to atarget. In some instances, a target comprises a target sequence for aprobe or probe set. In some instances, the plurality of probes or probesets may be used to generate a product comprising signal amplificationcomponents. In some instances, the amplification comprises one or moreprobe hybridizations and generation of amplified signals associated withthe labelling agents (e.g., probes). Exemplary signal amplificationmethods include targeted assembly of branched structures (e.g., bDNA).In some instances, detection of nucleic acids sequences in situ includescombination of the sequential decoding methods described herein with anassembly for branched signal amplification using the nucleic acid probesprovided herein. In some instances, the assembly complex comprises anamplifier hybridized directly or indirectly (via one or moreoligonucleotides) to a sequence of a cellular nucleic acid.

After contacting the biological sample with a plurality of labellingagents (e.g., probes or probe sets), the probes may be directly detectedby determining detectable labels (if present), and/or detected by usingone or more other probes that bind directly or indirectly to theplurality of probes or probe sets or products thereof. The one or moreother probes may comprise a detectable label. For instance, a primarynucleic acid probe can bind to a target nucleic acid in the sample, anda secondary nucleic acid probe can be introduced to bind to the primarynucleic acid probe, where the secondary nucleic acid probe or a productthereof can then be detected using detectable probes (e.g., detectablylabeled probes). Higher order probes that directly or indirectly bind tothe secondary nucleic acid probe or product thereof may also be used,and the higher order probes or products thereof can then be detectedusing detectably labeled probes.

In some instances, a secondary nucleic acid probe binds to a primarynucleic acid probe directly hybridized to the target nucleic acid. Asecondary nucleic acid probe (e.g., a first detectable probe or a seconddetectable probe disclosed herein) may contain a recognition sequenceable to bind to or hybridize with a primary nucleic acid probe (e.g.,probes or probe sets disclosed herein) or a product thereof (e.g., anRCA product), e.g., at a barcode sequence or portion(s) thereof of theprobes or probe sets or products thereof. In some embodiments, asecondary nucleic acid probe may bind to a combination of barcodesequences (which may be continuous or spaced from one another) in theprobes or probe sets, a product thereof. In some embodiments, thebinding is specific, or the binding may be such that a recognitionsequence preferentially binds to or hybridizes with only one of thebarcode sequences or complements thereof that are present. The secondarynucleic acid probe may also contain one or more detectable labels. Ifmore than one secondary nucleic acid probe is used, the detectablelabels may be the same or different.

The recognition sequences may be of any length, and multiple recognitionsequences in the same or different secondary nucleic acid probes may beof the same or different lengths. If more than one recognition sequenceis used, the recognition sequences may independently have the same ordifferent lengths. For instance, the recognition sequence may be atleast 4, at least 5, least 6, least 7, least 8, least 9, at least 10,least 11, least 12, least 13, least 14, at least 15, least 16, least 17,least 18, least 19, at least 20, at least 25, at least 30, at least 35,at least 40, or at least 50 nucleotides in length. In some embodiments,the recognition sequence may be no more than 48, no more than 40, nomore than 32, no more than 24, no more than 16, no more than 12, no morethan 10, no more than 8, or no more than 6 nucleotides in length.Combinations of any of these are also possible, e.g., the recognitionsequence may have a length of between 5 and 8, between 6 and 12, orbetween 7 and 15 nucleotides, etc. In some embodiments, the recognitionsequence is of the same length as a barcode sequence or complementthereof of a primary nucleic acid probe or a product thereof. In someembodiments, the recognition sequence may be at least 50%, at least 60%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 92%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or at least 100% complementary to the barcodesequence or complement thereof.

In some embodiments, the probes or probe sets, or an intermediate probe,may also comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ormore, 20 or more, 32 or more, 40 or more, or 50 or more barcodesequences. As an illustrative example, a first probe may contain a firsttarget-binding sequence, a first barcode sequence, and a second barcodesequence, while a second, different probe may contain a secondtarget-binding sequence (that is different from the first target-bindingsequence in the first probe), the same first barcode sequence as in thefirst probe, but a third barcode sequence instead of the second barcodesequence. Such probes may thereby be distinguished by determining thevarious barcode sequence combinations present or associated with a givenprobe at a given location in a sample.

In some embodiments, the nucleic acid probes disclosed herein may bemade using only 2 or only 3 of the 4 bases, such as leaving out all the“G” s and/or leaving out all of the “C” s within the probe. Sequenceslacking either “G” s or “C” s may form very little secondary structure,and can contribute to more uniform, faster hybridization in certainembodiments.

In some embodiments, a nucleic acid probe disclosed herein may contain adetectable label such as a fluorophore. In some embodiments, one or moreprobes of a plurality of nucleic acid probes used in an assay may lack adetectable label, while one or more other probes in the plurality eachcomprises a detectable label selected from a limited pool of distinctdetectable labels (e.g., red, green, yellow, and blue fluorophores), andthe absence of detectable label may be used as a separate “color.” Assuch, detectable labels are not required in all cases. In someembodiments, a primary nucleic acid probe disclosed herein lacks adetectable label. While a detectable label may be incorporated into anamplification product of a probe, such as via incorporation of amodified nucleotide into an RCA product of a circularized probe, theamplification product itself in some embodiments is not detectablylabeled. In some embodiments, a probe that binds to the primary nucleicacid probe or a product thereof (e.g., a secondary nucleic acid probethat binds to a barcode sequence or complement thereof in the primarynucleic acid probe or product thereof) comprises a detectable label andmay be used to detect the primary nucleic acid probe or product thereof.In some embodiments, a secondary nucleic acid probe disclosed hereinlacks a detectable label, and a detectably labeled probe that binds tothe secondary nucleic acid probe or a product thereof (e.g., at abarcode sequence or complement thereof in the secondary nucleic acidprobe or product thereof) can be used to detect the second nucleic acidprobe or product thereof. In some embodiments, signals associated withthe detectably labeled probes (e.g., the first detectable probe which isdetectably labelled, the second detectable probe which is detectablylabelled, a detectably labeled probe that binds to the first detectableprobe which itself is not detectably labelled, or a detectably labeledprobe that binds to the second detectable probe which itself is notdetectably labelled) can be used to detect one or more barcode sequencesin the secondary probe and/or one or more barcode sequences in theprimary probe, e.g., by using sequential hybridization of detectablylabeled probes, sequencing-by-ligation, and/orsequencing-by-hybridization. In some embodiments, the barcode sequences(e.g., in the secondary probe and/or in the primary probe) are used tocombinatorially encode a plurality of analytes of interest. As such,signals associated with the detectably labeled probes at particularlocations in a biological sample can be used to generate distinct signalsignatures that each corresponds to an analyte in the sample, therebyidentifying the analytes at the particular locations, e.g., for in situspatial analysis of the sample.

In some embodiments, probes or probe sets described herein comprises oneor more other components, such as one or more primer binding sequences(e.g., to allow for enzymatic amplification of probes), enzymerecognition sequences (e.g., for endonuclease cleavage), or the like.The components of the nucleic acid probe may be arranged in any suitableorder.

In some aspects, targets (e.g., analytes) are targeted by labellingagents (e.g., probes or probe sets) described herein, which are barcodedthrough the incorporation of one or more barcode sequences (e.g.,sequences that can be detected or otherwise “read”) and binds thetargeted analyte. In some aspects, the probes or probe sets describedherein are in turn targeted by secondary probes e.g., intermediateprobes, which are also barcoded through the incorporation of one or morebarcode sequences that are separate from a recognition sequence in asecondary probe that directly or indirectly binds the probes or probesets described herein or a product thereof. In some embodiments, asecondary probe may bind to a barcode sequence in the primary probe. Insome aspects, tertiary probes and optionally even higher order probesmay be used to target the secondary probes, e.g., at a barcode sequenceor complement thereof in a secondary probe or product thereof. In someembodiments, the tertiary probes and/or even higher order probes maycomprise one or more barcode sequences and/or one or more detectablelabels. In some embodiments, a tertiary probe is a detectably labeledprobe that hybridizes to a barcode sequence (or complement thereof) of asecondary probe (or product thereof). In some embodiments, through thedetection of signals associated with detectably labeled probes in asample, the location of one or more analytes in the sample and theidentity of the analyte(s) can be determined. In some embodiments, thepresence/absence, absolute or relative abundance, an amount, a level, aconcentration, an activity, and/or a relation with another analyte of aparticular analyte can be analyzed in situ in the sample.

In some embodiments, provided herein are labelling agents (e.g., probesor probe sets), and assay methods to couple target nucleic aciddetection, signal amplification (e.g., through nucleic acidamplification such as RCA, and/or hybridization of a plurality ofdetectably labeled probes, such as in hybridization chain reactions andthe like, e.g., described in Section III-C), and decoding of thebarcodes.

In some aspects, probes or probe sets described herein, or intermediateprobes (e.g., a secondary probe, and/or a higher order probe) can beselected from the group consisting of a circular probe, a circularizableprobe, and a linear probe. In some embodiments, a circular probe can beone that is pre-circularized prior to hybridization to a target nucleicacid and/or one or more other probes. In some embodiments, acircularizable probe can be one that can be circularized uponhybridization to a target nucleic acid and/or one or more other probessuch as a splint. In some embodiments, a linear probe can be one thatcomprises a target recognition sequence and a sequence that does nothybridize to a target nucleic acid, such as a 5′ overhang, a 3′overhang, and/or a linker or spacer (which may comprise a nucleic acidsequence or a non-nucleic acid moiety). In some embodiments, thesequence (e.g., the 5′ overhang, 3′ overhang, and/or linker or spacer)is non-hybridizing to the target nucleic acid but may hybridize to oneanother and/or one or more other probes, such as detectably labeledprobes.

Specific probe designs can vary depending on the application. Forinstance, probes or probe sets described herein (e.g., a primary probe,)or a secondary probe, and/or a higher order probe disclosed herein cancomprise a circularizable probe that does not require gap filling tocircularize upon hybridization to a template (e.g., a target nucleicacid and/or a probe such as a splint), a gapped circularizable probe(e.g., one that requires gap filling to circularize upon hybridizationto a template), an L-shaped probe (e.g., one that comprises a targetrecognition sequence and a 5′ or 3′ overhang upon hybridization to atarget nucleic acid or a probe), a U-shaped probe (e.g., one thatcomprises a target recognition sequence, a 5′ overhang, and a 3′overhang upon hybridization to a target nucleic acid or a probe), aV-shaped probe (e.g., one that comprises at least two target recognitionsequences and a linker or spacer between the target recognitionsequences upon hybridization to a target nucleic acid or a probe), aprobe or probe set for proximity ligation (such as those described inU.S. Pat. Nos. 7,914,987 and 8,580,504 incorporated herein by referencein their entireties, and probes for Proximity Ligation Assay (PLA) forthe simultaneous detection and quantification of nucleic acid moleculesand protein-protein interactions), or any suitable combination thereof.In some embodiments, a primary probe, a secondary probe, and/or a higherorder probe disclosed herein can comprise a probe that is ligated toitself or another probe using DNA-templated and/or RNA-templatedligation. In some embodiments, a primary probe, a secondary probe,and/or a higher order probe disclosed herein can be a DNA molecule andcan comprise one or more other types of nucleotides, modifiednucleotides, and/or nucleotide analogues, such as one or moreribonucleotides. In some embodiments, the ligation can be a DNA ligationon a DNA template. In some embodiments, the ligation can be a DNAligation on an RNA template, and the probes can comprise RNA-templatedligation probes. In some embodiments, a primary probe, a secondaryprobe, and/or a higher order probe disclosed herein can comprise apadlock-like probe or probe set, such as one described in US2019/0055594, US 2021/0164039, US 2016/0108458, or US 2020/0224243, eachof which is incorporated herein by reference in its entirety. Anysuitable combination of the probe designs described herein can be used.

In some embodiments, probes or probe sets described herein (e.g., aprimary probe,) or a secondary probe, and/or a higher order probedisclosed herein can comprise two or more parts. In some cases, a probecan comprise one or more features of and/or be modified based on: asplit FISH probe or probe set described in WO 2021/167526A1 or Goh etal., “Highly specific multiplexed RNA imaging in tissues withsplit-FISH,” Nat Methods 17(7):689-693 (2020), which are incorporatedherein by reference in their entireties; a Z-probe or probe set, such asone described in U.S. Pat. No. 7,709,198 B2, U.S. Pat. No. 8,604,182 B2,U.S. Pat. No. 8,951,726 B2, U.S. Pat. No. 8,658,361 B2, or Tripathi etal., “Z Probe, An Efficient Tool for Characterizing Long Non-Coding RNAin FFPE Tissues,” Noncoding RNA 4(3):20 (2018), which are incorporatedherein by reference in their entireties; an HCR initiator or amplifier,such as one described in U.S. Pat. No. 7,632,641 B2, US 2017/0009278 A1,U.S. Pat. No. 10,450,599 B2, Dirks and Pierce, “Triggered amplificationby hybridization chain reaction,” PNAS 101(43):15275-15278 (2004),Chemeris et al., “Real-time hybridization chain reaction,” Dokl. Biochem419:53-55 (2008), Niu et al., “Fluorescence detection for DNA usinghybridization chain reaction with enzyme-amplification,” Chem Commun(Camb) 46(18):3089-91 (2010), Choi et al., “Programmable in situamplification for multiplexed imaging of mRNA expression,” NatBiotechnol 28(11):1208-12 (2010), Song et al., “Hybridization chainreaction-based aptameric system for the highly selective and sensitivedetection of protein,” Analyst 137(6):1396-401 (2012), Choi et al.,“Third-generation in situ hybridization chain reaction: multiplexed,quantitative, sensitive, versatile, robust,” Development 145(12):dev165753 (2018), or Tsuneoka and Funato, “Modified in situHybridization Chain Reaction Using Short Hairpin DNAs,” Front MolNeurosci 13:75 (2020), which are incorporated herein by reference intheir entireties; a PLAYR probe or probe set, such as one described inUS 2016/0108458 A1 or Frei et al., “Highly multiplexed simultaneousdetection of RNAs and proteins in single cells,” Nat Methods13(3):269-75 (2016), which are incorporated herein by reference in theirentireties; a PLISH probe or probe set, such as one described in US2020/0224243 A1 or Nagendran et al., “Automated cell-type classificationin intact tissues by single-cell molecular profiling,” eLife 7:e30510(2018), which are incorporated herein by reference in their entireties;a RollFISH probe or probe set such as one described in Wu et al.,“RollFISH achieves robust quantification of single-molecule RNAbiomarkers in paraffin-embedded tumor tissue samples,” Commun Biol 1,209 (2018), which is hereby incorporated by reference in its entirety; aMERFISH probe or probe set, such as one described in US 2022/0064697 A1or Chen et al., “Spatially resolved, highly multiplexed RNA profiling insingle cells,” Science 348(6233):aaa6090 (2015), which are incorporatedherein by reference in their entireties; or a primer exchange reaction(PER) probe or probe set, such as one described in US 2019/0106733 A1,which is hereby incorporated by reference in its entirety.

In some embodiments, probes or probe sets described herein comprise oneor more features and/or is modified to allow for generation anddetection of a first signal that does not comprise a nucleic acidamplification step (e.g., the first signal can be an smFISH signal). Insome instances, the probes or probe sets described herein for eachtarget comprises probes directly hybridize to multiple regions (e.g.,sequences) of the same transcript. In some embodiments, the probes orprobe sets described herein comprise a circular probe or circularizableprobe or probe set comprises one or more features and/or is modified toallow for generation and detection of a second signal that comprises anamplification step (e.g., extension and/or amplification catalyzed by apolymerase).

Any suitable circularizable probe or probe set may be used to generatethe RCA template which is used to generate the RCA product. By“circularizable” is meant that the probe or reporter (the RCA template)is in the form of a linear molecule having ligatable ends which maycircularized by ligating the ends together directly or indirectly, e.g.to each other, or to the respective ends of an intervening (“gap”)oligonucleotide or to an extended 3′ end of the circularizable RCAtemplate. A circularizable template may also be provided in two or moreparts, namely two or more molecules (e.g. oligonucleotides) which may beligated together to form a circle. When said RCA template iscircularizable it is circularized by ligation prior to RCA. Ligation maybe templated using a ligation template. The circularizable RCA template(or template part or portion) may comprise at its respective 3′ and 5′ends regions of complementarity to corresponding cognate complementaryregions (or binding sites) in the ligation template, which may beadjacent where the ends are directly ligated to each other, ornon-adjacent, with an intervening “gap” sequence, where indirectligation is to take place.

In some embodiments, probes or probe sets disclosed herein can bepre-assembled from multiple components, e.g., prior to contacting theprobe with a target nucleic acid or a sample. In some embodiments, anucleic acid probe disclosed herein can be assembled during and/or aftercontacting a target nucleic acid or a sample with multiple components.In some embodiments, a nucleic acid probe disclosed herein is assembledin situ in a sample. In some embodiments, the multiple components can becontacted with a target nucleic acid or a sample in any suitable orderand any suitable combination. For instance, a first component and asecond component can be contacted with a target nucleic acid, to allowbinding between the components and/or binding between the first and/orsecond components with the target nucleic acid. Optionally a reactioninvolving either or both components and/or the target nucleic acid,between the components, and/or between either one or both components andthe target nucleic acid can be performed, such as hybridization,ligation, primer extension and/or amplification, chemical or enzymaticcleavage, click chemistry, or any combination thereof. In someembodiments, a third component can be added prior to, during, or afterthe reaction. In some embodiments, a third component can be added priorto, during, or after contacting the sample with the first and/or secondcomponents. In some embodiments, the first, second, and third componentscan be contacted with the sample in any suitable combination,sequentially or simultaneously. In some embodiments, the nucleic acidprobe can be assembled in situ in a stepwise manner, each step with theaddition of one or more components, or in a dynamic process where allcomponents are assembled together. One or more removing steps, e.g., bywashing the sample such as under stringent conditions, may be performedat any point during the assembling process to remove or destabilizeundesired intermediates and/or components at that point and increase thechance of accurate probe assembly and specific target binding of theassembled probe.

In some aspects, the methods provided herein comprise performing rollingcircle amplification of a circular probe or a circularized probegenerated from a circularizable probe or probe set.

In some embodiments, a probe disclosed herein can comprise a 5′ flapwhich may be recognized by a structure-specific cleavage enzyme, e.g. anenzyme capable of recognizing the junction between a single-stranded 5′overhang and a DNA duplex, and cleaving the single-stranded overhang. Itwill be understood that the branched three-strand structure which is thesubstrate for the structure-specific cleavage enzyme may be formed by 5′end of one probe part and the 3′ end of another probe part when bothhave hybridized to a target, as well as by the 5′ and 3′ ends of aone-part probe. Enzymes suitable for such cleavage include Flapendonucleases (FENS), which are a class of enzymes havingendonucleolytic activity and being capable of catalyzing the hydrolyticcleavage of the phosphodiester bond at the junction of single- anddouble-stranded DNA. Thus, in some embodiment, cleavage of theadditional sequence 5′ to the first target-specific binding site isperformed by a structure-specific cleavage enzyme, e.g. a Flapendonuclease. Suitable Flap endonucleases are described in Ma et al.2000. JBC 275, 24693-24700 and in US 2020/0224244 and may include P.furiosus (Pfu), A. fulgidus (Afu), M. jannaschii (Mja) or M.thermoautotrophicum (Mth). In other embodiments an enzyme capable ofrecognizing and degrading a single-stranded oligonucleotide having afree 5′ end may be used to cleave an additional sequence (5′ flap) froma structure as described above. Thus, an enzyme having 5′ nucleaseactivity may be used to cleave a 5′ additional sequence. Such 5′nuclease activity may be 5′ exonuclease and/or 5′ endonuclease activity.A 5′ nuclease enzyme is capable of recognizing a free 5′ end of asingle-stranded oligonucleotide and degrading said single-strandedoligonucleotide. A 5′ exonuclease degrades a single-strandedoligonucleotide having a free 5′ end by degrading the oligonucleotideinto constituent mononucleotides from its 5′ end. A 5′ endonucleaseactivity may cleave the 5′ flap sequence internally at one or morenucleotides. Further, a 5′ nuclease activity may take place by theenzyme traversing the single-stranded oligonucleotide to a region ofduplex once it has recognized the free 5′ end, and cleaving thesingle-stranded region into larger constituent nucleotides (e.g.dinucleotides or trinucleotides), or cleaving the entire 5′single-stranded region, e.g. as described in Lyamichev et al. 1999. PNAS96, 6143-6148 for Taq DNA polymerase and the 5′ nuclease thereof.Preferred enzymes having 5′ nuclease activity include Exonuclease VIII,or a native or recombinant DNA polymerase enzyme from Thermus aquaticus(Taq), Thermus thermophilus or Thermus flavus, or the nuclease domaintherefrom.

A target sequence for a probe disclosed herein may be comprised in anyanalyte (e.g., target) disclose herein, including an endogenous analyte(e.g., a viral or cellular nucleic acid), a labelling agent, or aproduct of an endogenous analyte and/or a labelling agent. In someembodiments, a target sequence for a probe disclosed herein comprisesone or more ribonucleotides.

In some aspects, one or more of the target sequences includes one ormore barcode(s), e.g., at least two, three, four, five, six, seven,eight, nine, ten, or more barcodes. Barcodes can spatially-resolvemolecular components found in biological samples, for example, within acell or a tissue sample. A barcode can be attached to an analyte or toanother moiety or structure in a reversible or irreversible manner. Abarcode can be added to, for example, a fragment of a deoxyribonucleicacid (DNA) or ribonucleic acid (RNA) sample before or during sequencingof the sample. Barcodes can allow for identification and/orquantification of individual sequencing-reads (e.g., a barcode can be orcan include a unique molecular identifier or “UMI”). In some aspects, abarcode comprises about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30nucleotides.

In some instances, a barcode may be a barcode region. In someembodiments, a barcode comprises two or more sub-barcodes that togetherfunction as a single barcode. For example, a polynucleotide barcode caninclude two or more polynucleotide sequences (e.g., sub-barcodes) thatare separated by one or more non-barcode sequences. In some embodiments,the one or more barcode(s) can also provide a platform for targetingfunctionalities, such as oligonucleotides, oligonucleotide-antibodyconjugates, oligonucleotide-streptavidin conjugates, modifiedoligonucleotides, affinity purification, detectable moieties, enzymes,enzymes for detection assays or other functionalities, and/or fordetection and identification of the polynucleotide.

In some embodiments, barcodes or complements thereof (e.g., barcodesequences or complements thereof comprised by the labelling agents(e.g., probes) disclosed herein or products thereof) can be analyzed(e.g., detected or sequenced) using any suitable method or technique,including those described herein, such as sequencing by synthesis (SBS),sequencing by ligation (SBL), or sequencing by hybridization (SBH). Insome instances, barcoding schemes and/or barcode detection schemes asdescribed in RNA sequential probing of targets (RNA SPOTs),single-molecule fluorescent in situ hybridization (smFISH), multiplexederror-robust fluorescence in situ hybridization (MERFISH) or sequentialfluorescence in situ hybridization (seqFISH+) can be used. In any of thepreceding implementations, the methods provided herein can includeanalyzing the barcodes by sequential hybridization and detection with aplurality of labelled probes (e.g., detection probes (e.g., detectionoligos) or barcode probes). In some instances, the barcode detectionsteps can be performed as described in hybridization-based in situsequencing (HybISS). In some instances, probes can be detected andanalyzed (e.g., detected or sequenced) as performed in fluorescent insitu sequencing (FISSEQ), or as performed in the detection steps of thespatially-resolved transcript amplicon readout mapping (STARmap) method.In some instances, signals associated with an analyte can be detected asperformed in sequential fluorescent in situ hybridization (seqFISH).

In some embodiments, in a barcode sequencing method, barcode sequencesare detected for identification of other molecules including nucleicacid molecules (DNA or RNA) longer than the barcode sequencesthemselves, as opposed to direct sequencing of the longer nucleic acidmolecules. In some embodiments, a N-mer barcode sequence comprises 4^(N)complexity given a sequencing read of N bases, and a much shortersequencing read may be required for molecular identification compared tonon-barcode sequencing methods such as direct sequencing. For example,1024 molecular species may be identified using a 5-nucleotide barcodesequence (4⁵=1024), whereas 8 nucleotide barcodes can be used toidentify up to 65,536 molecular species, a number greater than the totalnumber of distinct genes in the human genome. In some embodiments, thebarcode sequences contained in the probes or RCA products are detected,rather than endogenous sequences, which can be an efficient read-out interms of information per cycle of sequencing. Because the barcodesequences are pre-determined, they can also be designed to feature errordetection and correction mechanisms, see, e.g., U.S. Pat. Pub.20190055594 and U.S. Pat. Pub. 20210164039, which are herebyincorporated by reference in their entirety.

d. Detection

Provided herein are a plurality of detectable probes contacted with thebiological sample for detecting a plurality of signals associated with aplurality of targets in the biological sample. In some embodiments, thedetectable probes are detectably labeled or comprise a detectable label.The terms “label” and “detectable label” comprise a directly orindirectly detectable moiety that is associated with (e.g., conjugatedto) a molecule to be detected, e.g., a nucleic acid molecule thatcomprises a detectable label. The detectable label can be directlydetectable by itself (e.g., radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, can be indirectly detectable,e.g., by catalyzing chemical alterations of a substrate compound orcomposition, which substrate compound or composition is directlydetectable. Detectable labels can be suitable for small scale detectionand/or suitable for high-throughput screening. As such, suitabledetectable labels include, but are not limited to, radioisotopes(radioactive isotopes), fluorophores, fluorescers, chemiluminescentcompounds, bioluminescent compounds, dyes, enzymes, enzyme substrates,enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions,metal sols, ligands (e.g., biotin or haptens) and the like.

In some aspects, the detectable label comprises a luminophore. In someembodiments, the luminophore is a fluorophore. The term “fluorophore”comprises a substance or a portion thereof that is capable of exhibitingfluorescence in the detectable range. Particular examples of labels thatmay be used in accordance with the provided embodiments comprise, butare not limited to phycoerythrin, Alexa dyes (AlexaFluors), fluorescein,YPet, CyPet, Cascade blue, allophycocyanin, Cy3, Cy5, Cy7, rhodamine,dansyl, umbelliferone, Texas red, luminol, acradimum esters, biotin,green fluorescent protein (GFP), enhanced green fluorescent protein(EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescentprotein (EYFP), blue fluorescent protein (BFP), red fluorescent protein(RFP), firefly luciferase, Renilla luciferase, NADPH,beta-galactosidase, horseradish peroxidase, glucose oxidase, alkalinephosphatase, chloramphenical acetyl transferase, and urease.

In some embodiments, the detectable label is a fluorophore. For example,the fluorophore can be from a group that includes: 7-AAD(7-Aminoactinomycin D), Acridine Orange (+DNA), Acridine Orange (+RNA),Alexa Fluor® 350, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532,Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594,Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680,Alexa Fluor® 700, Alexa Fluor® 750, Allophycocyanin (APC), AMCA/AMCA-X,7-Aminoactinomycin D (7-AAD), 7-Amino-4-methylcoumarin,6-Aminoquinoline, Aniline Blue, ANS, APC-Cy7, ATTO-TAG™ CBQCA, ATTO-TAG™FQ, Auramine O-Feulgen, BCECF (high pH), BFP (Blue Fluorescent Protein),BFP/GFP FRET, BOBO™-1/BO-PRO™-1, BOBO™-3/BO-PRO™-3, BODIPY® FL, BODIPY®TMR, BODIPY® TR-X, BODIPY® 530/550, BODIPY® 558/568, BODIPY® 564/570,BODIPY® 581/591, BODIPY® 630/650-X, BODIPY® 650-665-X, BTC, Calcein,Calcein Blue, Calcium Crimson™, Calcium Green-1™, Calcium Orange™,Calcofluor® White, 5-Carboxyfluoroscein (5-FAM),5-Carboxynaphthofluoroscein, 6-Carboxyrhodamine 6G,5-Carboxytetramethylrhodamine (5-TAMRA), Carboxy-X-rhodamine (5-ROX),Cascade Blue®, Cascade Yellow™, CCF2 (GeneBLAzer™), CFP (CyanFluorescent Protein), CFP/YFP FRET, Chromomycin A3, Cl-NERF (low pH),CPM, 6-CR 6G, CTC Formazan, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy7®,Cychrome (PE-Cy5), Dansylamine, Dansyl cadaverine, Dansylchloride, DAPI,Dapoxyl, DCFH, DHR, DiA (4-Di-16-ASP), DiD (DilC18(5)), DIDS, Dil(DilC18(3)), DiO (DiOC18(3)), DiR (DilC18(7)), Di-4 ANEPPS, Di-8 ANEPPS,DM-NERF (4.5-6.5 pH), DsRed (Red Fluorescent Protein), EBFP, ECFP, EGFP,ELF®-97 alcohol, Eosin, Erythrosin, Ethidium bromide, Ethidiumhomodimer-1 (EthD-1), Europium (III) Chloride, 5-FAM(5-Carboxyfluorescein), Fast Blue, Fluorescein-dT phosphoramidite, FITC,Fluo-3, Fluo-4, FluorX®, Fluoro-Gold™ (high pH), Fluoro-Gold™ (low pH),Fluoro-Jade, FM® 1-43, Fura-2 (high calcium), Fura-2/BCECF, Fura Red™(high calcium), Fura Red™/Fluo-3, GeneBLAzer™ (CCF2), GFP Red Shifted(rsGFP), GFP Wild Type, GFP/BFP FRET, GFP/DsRed FRET, Hoechst 33342 &33258, 7-Hydroxy-4-methylcoumarin (pH 9), 1,5 IAEDANS, Indo-1 (highcalcium), Indo-1 (low calcium), Indodicarbocyanine, Indotricarbocyanine,JC-1,6-JOE, JOJO™-1/JO-PRO™-1, LDS 751 (+DNA), LDS 751 (+RNA),LOLO™-1/LO-PRO™-1, Lucifer Yellow, LysoSensor™ Blue (pH 5), LysoSensor™Green (pH 5), LysoSensor™ Yellow/Blue (pH 4.2), LysoTracker® Green,LysoTracker® Red, LysoTracker® Yellow, Mag-Fura-2, Mag-Indo-1, MagnesiumGreen™, Marina Blue®, 4-Methylumbelliferone, Mithramycin, MitoTracker®Green, MitoTracker® Orange, MitoTracker® Red, NBD (amine), Nile Red,Oregon Green® 488, Oregon Green® 500, Oregon Green® 514, Pacific Blue,PBF1, PE (R-phycoerythrin), PE-Cy5, PE-Cy7, PE-Texas Red, PerCP(Peridinin chlorphyll protein), PerCP-Cy5.5 (TruRed), PharRed (APC-Cy7),C-phycocyanin, R-phycocyanin, R-phycoerythrin (PE), PI (PropidiumIodide), PKH26, PKH67, POPO™-1/PO-PRO™-1, POPO™-3/PO-PRO™-3, PropidiumIodide (PI), PyMPO, Pyrene, Pyronin Y, Quantam Red (PE-Cy5), QuinacrineMustard, R670 (PE-Cy5), Red 613 (PE-Texas Red), Red Fluorescent Protein(DsRed), Resorufin, RH 414, Rhod-2, Rhodamine B, Rhodamine Green™,Rhodamine Red™, Rhodamine Phalloidin, Rhodamine 110, Rhodamine 123,5-ROX (carboxy-X-rhodamine), S65A, S65C, S65L, S65T, SBFI, SITS,SNAFL®-1 (high pH), SNAFL®-2, SNARF®-1 (high pH), SNARF®-1 (low pH),Sodium Green™, SpectrumAqua®, SpectrumGreen® #1, SpectrumGreen® #2,SpectrumOrange®, SpectrumRed®, SYTO® 11, SYTO® 13, SYTO® 17, SYTO® 45,SYTOX® Blue, SYTOX® Green, SYTOX® Orange, 5-TAMRA(5-Carboxytetramethylrhodamine), Tetramethylrhodamine (TRITC), TexasRed®/Texas Red®-X, Texas Red®-X (NHS Ester), Thiadicarbocyanine,Thiazole Orange, TOTO®-1/TO-PRO®-1, TOTO®-3/TO-PRO®-3, TO-PRO®-5,Tri-color (PE-Cy5), TRITC (Tetramethylrhodamine), TruRed (PerCP-Cy5.5),WW 781, X-Rhodamine (XRITC), Y66F, Y66H, Y66W, YFP (Yellow FluorescentProtein), YOYO®-1/YO-PRO®-1, YOYO®-3/YO-PRO®-3,6-FAM (Fluorescein),6-FAM (NHS Ester), 6-FAM (Azide), HEX, TAMRA (NHS Ester), Yakima Yellow,MAX, TET, TEX615, ATTO 488, ATTO 532, ATTO 550, ATTO 565, ATTO Rhol01,ATTO 590, ATTO 633, ATTO 647N, TYE 563, TYE 665, TYE 705, 5′ IRDye® 700,5′ IRDye® 800, 5′ IRDye® 800CW (NHS Ester), WellRED D4 Dye, WellRED D3Dye, WellRED D2 Dye, Lightcycler® 640 (NHS Ester), and Dy 750 (NHSEster).

In some embodiments, the detectable label comprises an infraredfluorophore. An “infrared fluorophore” emits infrared light. In someembodiments, the infrared fluorophore has a longer excitation wavelengththan a traditional fluorophore.

Examples of detectable labels comprise, but are not limited to, variousradioactive moieties, enzymes, prosthetic groups, fluorescent markers,luminescent markers, bioluminescent markers, metal particles,protein-protein binding pairs and protein-antibody binding pairs.Examples of fluorescent proteins comprise, but are not limited to,yellow fluorescent protein (YFP), green fluorescence protein (GFP), cyanfluorescence protein (CFP), umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride and phycoerythrin.

Examples of bioluminescent markers comprise, but are not limited to,luciferase (e.g., bacterial, firefly and click beetle), luciferin,aequorin and the like. Examples of enzyme systems having visuallydetectable signals comprise, but are not limited to, galactosidases,glucorimidases, phosphatases, peroxidases and cholinesterases.Identifiable markers also comprise radioactive compounds such as ¹²⁵I,³⁵S, ¹⁴C, or ³H. Identifiable markers are commercially available from avariety of sources.

Examples of fluorescent labels and nucleotides and/or polynucleotidesconjugated to such fluorescent labels comprise those described in, forexample, Hoagland, Handbook of Fluorescent Probes and ResearchChemicals, Ninth Edition (Molecular Probes, Inc., Eugene, 2002); Kellerand Manak, DNA Probes, 2nd Edition (Stockton Press, New York, 1993);Eckstein, editor, Oligonucleotides and Analogues: A Practical Approach(IRL Press, Oxford, 1991); and Wetmur, Critical Reviews in Biochemistryand Molecular Biology, 26:227-259 (1991). In some embodiments, exemplarytechniques and methods methodologies applicable to the providedembodiments comprise those described in, for example, U.S. Pat. Nos.4,757,141, 5,151,507 and 5,091,519. In some embodiments, one or morefluorescent dyes are used as labels for labeled target sequences, forexample, as described in U.S. Pat. No. 5,188,934(4,7-dichlorofluorescein dyes); U.S. Pat. No. 5,366,860 (spectrallyresolvable rhodamine dyes); U.S. Pat. No. 5,847,162(4,7-dichlororhodamine dyes); U.S. Pat. No. 4,318,846 (ether-substitutedfluorescein dyes); U.S. Pat. No. 5,800,996 (energy transfer dyes); U.S.Pat. No. 5,066,580 (xanthine dyes); and U.S. Pat. No. 5,688,648 (energytransfer dyes). Labelling can also be carried out with quantum dots, asdescribed in U.S. Pat. Nos. 6,322,901, 6,576,291, 6,423,551, 6,251,303,6,319,426, 6,426,513, 6,444,143, 5,990,479, 6,207,392, US 2002/0045045and US 2003/0017264. As used herein, the term “fluorescent label”comprises a signaling moiety that conveys information through thefluorescent absorption and/or emission properties of one or moremolecules. Exemplary fluorescent properties comprise fluorescenceintensity, fluorescence lifetime, emission spectrum characteristics andenergy transfer.

In some embodiments, one or more detectable labels can be attached to alabelling agent or nucleic acid probe disclosed herein. The one or moredetectable labels can be incorporated during nucleic acid polymerizationor amplification (e.g., Cy5®-labeled nucleotides, such as Cy5®-dCTP).Examples of commercially available fluorescent nucleotide analoguesreadily incorporated into nucleotide and/or polynucleotide sequencescomprise, but are not limited to, Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP(Amersham Biosciences, Piscataway, N.J.), fluorescein-12-dUTP,tetramethylrhodamine-6-dUTP, TEXAS RED™-5-dUTP, CASCADE BLUE™-7-dUTP,BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY TMTR-14-dUTP, RHOD AMINEGREEN™-5-dUTP, OREGON GREENR™ 488-5-dUTP, TEXAS RED™-12-dUTP, BODIPY™630/650-14-dUTP, BODIPY™ 650/665-14-dUTP, ALEXA FLUOR™ 488-5-dUTP, ALEXAFLUOR™ 532-5-dUTP, ALEXA FLUOR™ 568-5-dUTP, ALEXA FLUOR™ 594-5-dUTP,ALEXA FLUOR™ 546-14-dUTP, fluorescein-12-UTP,tetramethylrhodamine-6-UTP, TEXAS RED™-5-UTP, mCherry, CASCADEBLUE™-7-UTP, BODIPY™ FL-14-UTP, BODIPY TMR-14-UTP, BODIPY™ TR-14-UTP,RHOD AMINE GREEN™-5-UTP, ALEXA FLUOR™ 488-5-UTP, and ALEXA FLUOR™546-14-UTP (Molecular Probes, Inc. Eugene, Oreg.). Methods for customsynthesis of nucleotides having other fluorophores can include thosedescribed in Henegariu et a1. (2000) Nature Biotechnol. 18:345,incorporated herein by reference.

In some embodiments, one or more detectable labels can be attached viapost-synthetic attachment. Fluorophores available for post-syntheticattachment comprise, but are not limited to, ALEXA FLUOR™ 350, ALEXAFLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 568, ALEXA FLUOR™ 594, ALEXAFLUOR™ 647, BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY 530/550,BODIPY TMR, BODIPY 558/568, BODIPY 558/568, BODIPY 564/570, BODIPY576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665, Cascade Blue,Cascade Yellow, Dansyl, lissamine rhodamine B, Marina Blue, Oregon Green488, Oregon Green 514, Pacific Blue, rhodamine 6G, rhodamine green,rhodamine red, tetramethyl rhodamine, Texas Red (available fromMolecular Probes, Inc., Eugene, Oreg.), Cy2, Cy3.5, Cy5.5, and Cy7(Amersham Biosciences, Piscataway, N.J.). FRET tandem fluorophores mayalso be used, comprising, but not limited to, PerCP-Cy5.5, PE-Cy5,PE-Cy5.5, PE-Cy7, PE-Texas Red, APC-Cy7, PE-Alexa dyes (610, 647, 680),and APC-Alexa dyes.

In some cases, metallic silver or gold particles may be used to enhancesignal from fluorescently labeled nucleotide and/or polynucleotidesequences (Lakowicz et a1. (2003) Bio Techniques 34:62)

Biotin, or a derivative thereof, may also be used as a label on anucleic acid molecule, and subsequently bound by a detectably labeledavidin/streptavidin derivative (e.g., phycoerythrin-conjugatedstreptavidin), or a detectably labeled anti-biotin antibody. Digoxigeninmay be incorporated as a label and subsequently bound by a detectablylabeled anti-digoxigenin antibody (e.g., fluoresceinatedanti-digoxigenin). An aminoallyl-dUTP residue may be incorporated into apolynucleotide sequence and subsequently coupled to an N-hydroxysuccinimide (NHS) derivatized fluorescent dye. In general, any member ofa conjugate pair may be incorporated into a detection polynucleotideprovided that a detectably labeled conjugate partner can be bound topermit detection. As used herein, the term antibody refers to anantibody molecule of any class, or any sub-fragment thereof, such as anFab.

Other suitable labels for use in the methods provided herein maycomprise fluorescein (FAM), digoxigenin, dinitrophenol (DNP), dansyl,biotin, bromodeoxyuridine (BrdU), hexahistidine (6xHis), andphosphor-amino acids (e.g., P-tyr, P-ser, P-thr). In some embodimentsthe following hapten/antibody pairs are used for detection, in whicheach of the antibodies is derivatized with a detectable label:biotin/a-biotin, digoxigenin/a-digoxigenin, dinitrophenol (DNP)/a-DNP,5-Carboxyfluorescein (FAM)/a-FAM.

In some embodiments, a nucleic acid molecule (e.g., detectable probe)can be indirectly labeled, especially with a hapten that is then boundby a capture agent, e.g., as disclosed in U.S. Pat. Nos. 5,344,757,5,702,888, 5,354,657, 5,198,537 and 4,849,336, and 5,073,562, each ofwhich is herein incorporated by reference in its entirety. Manydifferent hapten-capture agent pairs are available for use. Exemplaryhaptens comprise, but are not limited to, biotin, des-biotin and otherderivatives, dinitrophenol, dansyl, fluorescein, Cy5, and digoxigenin.For biotin, a capture agent may be avidin, streptavidin, or antibodies.Antibodies may be used as capture agents for the other haptens (manydye-antibody pairs being commercially available, e.g., Molecular Probes,Eugene, Oreg.).

In some embodiments, a detectable label is or includes a luminescent orchemiluminescent moiety. Common luminescent/chemiluminescent moietiesinclude, but are not limited to, peroxidases such as horseradishperoxidase (HRP), soybean peroxidase (SP), alkaline phosphatase, andluciferase. These protein moieties can catalyze chemiluminescentreactions given the appropriate substrates (e.g., an oxidizing reagentplus a chemiluminescent compound. Non-limiting examples ofchemiluminescent compound families include2,3-dihydro-1,4-phthalazinedione luminol, 5-amino-6,7,8-trimethoxy- andthe dimethylamino[ca]benz analog. These compounds can luminesce in thepresence of alkaline hydrogen peroxide or calcium hypochlorite and base.Other examples of chemiluminescent compound families include, e.g.,2,4,5-triphenylimidazoles, para-dimethylamino and -methoxy substituents,oxalates such as oxalyl active esters, p-nitrophenyl, N-alkyl acridinumesters, luciferins, lucigenins, or acridinium esters. In someembodiments, a detectable label is or includes a metal-based ormass-based label. For example, small cluster metal ions, metals, orsemiconductors may act as a mass code. In some examples, the metals canbe selected from Groups 3-15 of the periodic table, e.g., Y, La, Ag, Au,Pt, Ni, Pd, Rh, Ir, Co, Cu, Bi, or a combination thereof.

In some embodiments, the detectable label is detected in situ. Thedetectable label can be qualitatively detected (e.g., optically orspectrally), or it can be quantified. Qualitative detection generallyincludes a detection method in which the existence or presence of thedetectable label is confirmed, whereas quantifiable detection generallyincludes a detection method having a quantifiable (e.g., numericallyreportable) value such as an intensity, duration, polarization, and/orother properties. For example, detectably labeled features can include afluorescent, a colorimetric, or a chemiluminescent label attached to abead (see, for example, Rajeswari et al., J. Microbiol Methods139:22-28, 2017, and Forcucci et al., J. Biomed Opt. 10:105010, 2015,the entire contents of each of which are incorporated herein byreference).

In some aspects, the method includes detection of the probe or probe sethybridized to the target (e.g., target sequence) or any productsgenerated therefrom or a derivative thereof. In any of the embodimentsherein, the method can further comprise imaging the biological sample todetect a ligation product or a circularized probe or product thereof. Inany of the embodiments herein, a sequence of the ligation product,rolling circle amplification product, or other generated product can beanalyzed in situ in the biological sample. In any of the embodimentsherein, the imaging can comprise detecting a signal associated with afluorescently labeled probe that directly or indirectly binds to arolling circle amplification product of the circularized probe. In anyof the embodiments herein, the sequence of the sequence of the ligationproduct, rolling circle amplification product, or other generatedproduct can be analyzed by sequential hybridization, sequencing byhybridization, sequencing by ligation, sequencing by synthesis,sequencing by binding, or a combination thereof.

In any of the embodiments herein, a sequence associated with the targetnucleic acid or the probes or probe sets described herein can compriseone or more barcode sequences or complements thereof. In any of theembodiments herein, the sequence of the rolling circle amplificationproduct can comprise one or more barcode sequences or complementsthereof. In any of the embodiments herein, a probe can comprise one ormore barcode sequences or complements thereof. In any of the embodimentsherein, the one or more barcode sequences can comprise a barcodesequence corresponding to the target nucleic acid. In any of theembodiments herein, the one or more barcode sequences can comprise abarcode sequence corresponding to the sequence of interest, such asvariant(s) of a single nucleotide of interest.

In any of the embodiments herein, the detecting step can comprisecontacting the biological sample with one or more detectable probes thatdirectly or indirectly hybridize to the rolling circle amplificationproduct, and dehybridizing the one or more detectable probes from therolling circle amplification product. In any of the embodiments herein,the contacting and dehybridizing steps can be repeated with the one ormore detectable probes and/or one or more other detectable probes thatdirectly or indirectly hybridize to the rolling circle amplificationproduct.

In any of the embodiments herein, the detecting step can comprisecontacting the biological sample with one or more first detectableprobes that directly hybridize to the plurality of probes or probe sets.In some instances, the detecting step can comprise contacting thebiological sample with one or more first detectable probes thatindirectly hybridize to the plurality of probes or probe sets. In any ofthe embodiments herein, the detecting step can comprise contacting thebiological sample with one or more first detectable probes that directlyor indirectly hybridize to the plurality of probes or probe sets.

In any of the embodiments herein, the detecting step can comprisecontacting the biological sample with one or more intermediate probesthat directly or indirectly hybridize to the plurality of probes orprobe sets, rolling circle amplification product generated using theplurality of probes or probe sets, wherein the one or more intermediateprobes are detectable using one or more detectable probes. In any of theembodiments herein, the detecting step can further comprisedehybridizing the one or more intermediate probes and/or the one or moredetectable probes from the rolling circle amplification product or theplurality of probes or probe sets. In any of the embodiments herein, thecontacting and dehybridizing steps can be repeated with the one or moreintermediate probes, the one or more detectable probes, one or moreother intermediate probes, and/or one or more other detectable probes.

In some embodiments, the detection may be spatial, e.g., in two or threedimensions. In some embodiments, the detection may be quantitative,e.g., the amount or concentration of a primary nucleic acid probe (andof a target nucleic acid) may be determined. In some embodiments, theplurality of probes or probe sets (e.g., primary probes), secondaryprobes, higher order probes, and/or detectable probes may comprise anyof a variety of entities able to hybridize a nucleic acid, e.g., DNA,RNA, LNA, and/or PNA, etc., depending on the application.

In some embodiments, a method disclosed herein may also comprise one ormore signal amplification components. In some embodiments, the presentdisclosure relates to the detection of nucleic acids sequences in situusing probe hybridization and generation of amplified signals associatedwith the probes, wherein background signal is reduced and sensitivity isincreased. In some embodiments, the RCA product generated using a methoddisclosed herein can be detected in with a method that comprises signalamplification. In some embodiments, signal amplification may compriseuse of the plurality of probes or probe sets.

Exemplary signal amplification methods include targeted deposition ofdetectable reactive molecules around the site of probe hybridization,targeted assembly of branched structures (e.g., bDNA or branched assayusing locked nucleic acid (LNA)), programmed in situ growth ofconcatemers by enzymatic rolling circle amplification (RCA) (e.g., asdescribed in US 2019/0055594 incorporated herein by reference),hybridization chain reaction, assembly of topologically catenated DNAstructures using serial rounds of chemical ligation (clampFISH), signalamplification via hairpin-mediated concatemerization (e.g., as describedin US 2020/0362398 incorporated herein by reference), e.g., primerexchange reactions such as signal amplification by exchange reaction(SABER) or SABER with DNA-Exchange (Exchange-SABER). In someembodiments, a non-enzymatic signal amplification method may be used.

The detectable reactive molecules may comprise tyramide, such as used intyramide signal amplification (TSA) or multiplexed catalyzed reporterdeposition (CARD)-FISH. In some embodiments, the detectable reactivemolecule may be releasable and/or cleavable from a detectable label suchas a fluorophore. In some embodiments, a method disclosed hereincomprises multiplexed analysis of a biological sample comprisingconsecutive cycles of probe hybridization, fluorescence imaging, andsignal removal, where the signal removal comprises removing thefluorophore from a fluorophore-labeled reactive molecule (e.g.,tyramide). Exemplary detectable reactive reagents and methods aredescribed in U.S. Pat. No. 6,828,109, US 2019/0376956, US 2019/0376956,US 2022/0026433, US 2022/0128565, and US 2021/0222234, all of which areincorporated herein by reference in their entireties.

In some embodiments, hybridization chain reaction (HCR) can be used forsignal amplification. HCR is an enzyme-free nucleic acid amplificationbased on a triggered chain of hybridization of nucleic acid moleculesstarting from HCR monomers, which hybridize to one another to form anicked nucleic acid polymer. This polymer is the product of the HCRreaction which is ultimately detected in order to indicate the presenceof the target analyte. HCR is described in detail in Dirks and Pierce,2004, PNAS, 101(43), 15275-15278 and in U.S. Pat. Nos. 7,632,641 and7,721,721 (see also US 2006/00234261; Chemeris et a1, 2008 DokladyBiochemistry and Biophysics, 419, 53-55; Niu et a1, 2010, 46, 3089-3091;Choi et a1, 2010, Nat. Biotechnol. 28(11), 1208-1212; and Song et a1,2012, Analyst, 137, 1396-1401). HCR monomers typically comprise ahairpin, or other metastable nucleic acid structure. In the simplestform of HCR, two different types of stable hairpin monomer, referred tohere as first and second HCR monomers, undergo a chain reaction ofhybridization events to form a long nicked double-stranded DNA moleculewhen an “initiator” nucleic acid molecule is introduced. The HCRmonomers have a hairpin structure comprising a double stranded stemregion, a loop region connecting the two strands of the stem region, anda single stranded region at one end of the double stranded stem region.The single stranded region which is exposed (and which is thus availablefor hybridization to another molecule, e.g. initiator or other HCRmonomer) when the monomers are in the hairpin structure may be known asthe “toehold region” (or “input domain”). The first HCR monomers eachfurther comprise a sequence which is complementary to a sequence in theexposed toehold region of the second HCR monomers. This sequence ofcomplementarity in the first HCR monomers may be known as the“interacting region” (or “output domain”). Similarly, the second HCRmonomers each comprise an interacting region (output domain), e.g. asequence which is complementary to the exposed toehold region (inputdomain) of the first HCR monomers. In the absence of the HCR initiator,these interacting regions are protected by the secondary structure (e.g.they are not exposed), and thus the hairpin monomers are stable orkinetically trapped (also referred to as “metastable”), and remain asmonomers (e.g. preventing the system from rapidly equilibrating),because the first and second sets of HCR monomers cannot hybridize toeach other. However, once the initiator is introduced, it is able tohybridize to the exposed toehold region of a first HCR monomer, andinvade it, causing it to open up. This exposes the interacting region ofthe first HCR monomer (e.g. the sequence of complementarity to thetoehold region of the second HCR monomers), allowing it to hybridize toand invade a second HCR monomer at the toehold region. Thishybridization and invasion in turn opens up the second HCR monomer,exposing its interacting region (which is complementary to the toeholdregion of the first HCR monomers), and allowing it to hybridize to andinvade another first HCR monomer. The reaction continues in this manneruntil all of the HCR monomers are exhausted (e.g. all of the HCRmonomers are incorporated into a polymeric chain). Ultimately, thischain reaction leads to the formation of a nicked chain of alternatingunits of the first and second monomer species. The presence of the HCRinitiator is thus required in order to trigger the HCR reaction byhybridization to and invasion of a first HCR monomer. The first andsecond HCR monomers are designed to hybridize to one another are thusmay be defined as cognate to one another. They are also cognate to agiven HCR initiator sequence. HCR monomers which interact with oneanother (hybridize) may be described as a set of HCR monomers or an HCRmonomer, or hairpin, system.

An HCR reaction could be carried out with more than two species or typesof HCR monomers. For example, a system involving three HCR monomerscould be used. In such a system, each first HCR monomer may comprise aninteracting region which binds to the toehold region of a second HCRmonomer; each second HCR may comprise an interacting region which bindsto the toehold region of a third HCR monomer; and each third HCR monomermay comprise an interacting region which binds to the toehold region ofa first HCR monomer. The HCR polymerization reaction would then proceedas described above, except that the resulting product would be a polymerhaving a repeating unit of first, second and third monomersconsecutively. Corresponding systems with larger numbers of sets of HCRmonomers could readily be conceived.

In some embodiments, similar to HCR reactions that use hairpin monomers,linear oligo hybridization chain reaction (LO-HCR) can also be used forsignal amplification. In some embodiments, provided herein is a methodof detecting an analyte in a sample comprising: (i) performing a linearoligo hybridization chain reaction (LO-HCR), wherein an initiator iscontacted with a plurality of LO-HCR monomers of at least a first and asecond species to generate a polymeric LO-HCR product hybridized to atarget nucleic acid molecule, wherein the first species comprises afirst hybridization region complementary to the initiator and a secondhybridization region complementary to the second species, wherein thefirst species and the second species are linear, single-stranded nucleicacid molecules; wherein the initiator is provided in one or more parts,and hybridizes directly or indirectly to or is comprised in the targetnucleic acid molecule; and (ii) detecting the polymeric product, therebydetecting the analyte. In some embodiments, the first species and/or thesecond species may not comprise a hairpin structure. In someembodiments, the plurality of LO-HCR monomers may not comprise ametastable secondary structure. In some embodiments, the LO-HCR polymermay not comprise a branched structure. In some embodiments, performingthe linear oligo hybridization chain reaction comprises contacting thetarget nucleic acid molecule with the initiator to provide the initiatorhybridized to the target nucleic acid molecule. In any of theembodiments herein, the target nucleic acid molecule and/or the analytecan be an RCA product. Exemplary methods and compositions for LO-HCR aredescribed in US 2021/0198723, incorporated herein by reference in itsentirety.

In some embodiments, detection of nucleic acids sequences in situ maycomprise an assembly for branched signal amplification. In someembodiments, the assembly complex comprises an amplifier hybridizeddirectly or indirectly (via one or more oligonucleotides) to a probe orprobe set. In some embodiments, the assembly includes one or moreamplifiers each including an amplifier repeating sequence. In someaspects, the one or more amplifiers is labeled. Described herein is amethod of using the aforementioned assembly, including for example,using the assembly in multiplexed error-robust fluorescent in situhybridization (MERFISH) applications, with branched DNA amplificationfor signal readout. In some embodiments, the amplifier repeatingsequence is about 5-30 nucleotides, and is repeated N times in theamplifier. In some embodiments, the amplifier repeating sequence isabout 20 nucleotides, and is repeated at least two times in theamplifier. In some aspects, the one or more amplifier repeating sequenceis labeled. For exemplary branched signal amplification, see e.g., U.S.Pat. Pub. No. US20200399689A1 and Xia et al., Multiplexed Detection ofRNA using MERFISH and branched DNA amplification. Scientific Reports(2019), each of which is fully incorporated by reference herein.

In some embodiments, the plurality of probes or probe sets can bedetected in with a method that comprises signal amplification byperforming a primer exchange reaction (PER). In various embodiments, aprimer with domain on its 3′ end binds to a catalytic hairpin, and isextended with a new domain by a strand displacing polymerase. Forexample, a primer with domain 1 on its 3′ ends binds to a catalytichairpin, and is extended with a new domain 1 by a strand displacingpolymerase, with repeated cycles generating a concatemer of repeateddomain 1 sequences. In various embodiments, the strand displacingpolymerase is Bst. In various embodiments, the catalytic hairpinincludes a stopper which releases the strand displacing polymerase. Invarious embodiments, branch migration displaces the extended primer,which can then dissociate. In various embodiments, the primer undergoesrepeated cycles to form a concatemer primer. In various embodiments, aplurality of concatemer primers is contacted with a sample comprisingthe plurality of probes or probe sets described herein. In variousembodiments, the plurality of probes or probe sets may be contacted witha plurality of concatemer primers and a plurality of labeled probes. seee.g., U.S. Pat. Pub. No. US20190106733, which is incorporated herein byreference, for exemplary molecules and PER reaction components.

In some embodiments, the methods comprise sequencing all or a portion ofthe amplification product, such as one or more barcode sequences presentin the amplification product, e.g., via DNA sequencing.

In some embodiments, the analysis and/or sequence determinationcomprises sequencing all or a portion of the amplification product orthe probe(s) and/or in situ hybridization to the amplification productor the probe(s). In some embodiments, the sequencing step involvessequencing by hybridization, sequencing by ligation, and/or fluorescentin situ sequencing, hybridization-based in situ sequencing and/orwherein the in situ hybridization comprises sequential fluorescent insitu hybridization. In some embodiments, the analysis and/or sequencedetermination comprises detecting a polymer generated by a hybridizationchain reaction (HCR) reaction, see e.g., US 2017/0009278, which isincorporated herein by reference, for exemplary probes and HCR reactioncomponents. In some embodiments, the detection or determinationcomprises hybridizing to the amplification product a detectionoligonucleotide labeled with a fluorophore, an isotope, a mass tag, or acombination thereof. In some embodiments, the detection or determinationcomprises imaging the amplification product. In some embodiments, thetarget nucleic acid is an mRNA in a tissue sample, and the detection ordetermination is performed when the target nucleic acid and/or theamplification product is in situ in the tissue sample.

In some aspects, the provided methods comprise imaging the amplificationproduct (e.g., amplicon) and/or one or more portions of the plurality ofprobes or probe sets, for example, via binding of the detection probeand detecting the detectable label. In some embodiments, the detectionprobe comprises a detectable label that can be measured and quantitated.The terms “label” and “detectable label” comprise a directly orindirectly detectable moiety that is associated with (e.g., conjugatedto) a molecule to be detected, e.g., a detectable probe, comprising, butnot limited to, fluorophores, radioactive isotopes, fluorescers,chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzymeinhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g.,biotin or haptens) and the like.

The term “fluorophore” comprises a substance or a portion thereof thatis capable of exhibiting fluorescence in the detectable range.Particular examples of labels that may be used in accordance with theprovided embodiments comprise, but are not limited to phycoerythrin,Alexa dyes, fluorescein, YPet, CyPet, Cascade blue, allophycocyanin,Cy3, Cy5, Cy7, rhodamine, dansyl, umbelliferone, Texas red, luminol,acradimum esters, biotin, green fluorescent protein (GFP), enhancedgreen fluorescent protein (EGFP), yellow fluorescent protein (YFP),enhanced yellow fluorescent protein (EYFP), blue fluorescent protein(BFP), red fluorescent protein (RFP), firefly luciferase, Renillaluciferase, NADPH, beta-galactosidase, horseradish peroxidase, glucoseoxidase, alkaline phosphatase, chloramphenical acetyl transferase, andurease.

Fluorescence detection in tissue samples can often be hindered by thepresence of strong background fluorescence. “Autofluorescence” is thegeneral term used to distinguish background fluorescence (that can arisefrom a variety of sources, including aldehyde fixation, extracellularmatrix components, red blood cells, lipofuscin, and the like) from thedesired immunofluorescence from the fluorescently labeled antibodies orprobes. Tissue autofluorescence can lead to difficulties indistinguishing the signals due to fluorescent antibodies or probes fromthe general background. In some embodiments, a method disclosed hereinutilizes one or more agents to reduce tissue autofluorescence, forexample, Autofluorescence Eliminator (Sigma/EMD Millipore), TrueBlackLipofuscin Autofluorescence Quencher (Biotium), MaxBlockAutofluorescence Reducing Reagent Kit (MaxVision Biosciences), and/or avery intense black dye (e.g., Sudan Black, or comparable darkchromophore).

In some embodiments, a detectable probe containing a detectable labelcan be used to detect the plurality of probes or probe sets and/oramplification products (e.g., amplicon) described herein. In someembodiments, the methods involve incubating the detectable probecontaining the detectable label with the sample, washing unbounddetectable probe, and detecting the label, e.g., by imaging.

In some aspects, the detecting comprises performing microscopy, scanningmass spectrometry or other imaging techniques described herein. In suchaspects, the detecting comprises determining a signal, e.g., afluorescent signal. In some aspects, the detection (comprising imaging)is carried out using any of a number of different types of microscopy,e.g., confocal microscopy, two-photon microscopy, light-fieldmicroscopy, intact tissue expansion microscopy, and/orCLARITY™-optimized light sheet microscopy (COLM).

In some embodiments, fluorescence microscopy is used for detection andimaging of the detection probe. In some aspects, a fluorescencemicroscope is an optical microscope that uses fluorescence andphosphorescence instead of, or in addition to, reflection and absorptionto study properties of organic or inorganic substances. In fluorescencemicroscopy, a sample is illuminated with light of a wavelength whichexcites fluorescence in the sample. The fluoresced light, which isusually at a longer wavelength than the illumination, is then imagedthrough a microscope objective. Two filters may be used in thistechnique; an illumination (or excitation) filter which ensures theillumination is near monochromatic and at the correct wavelength, and asecond emission (or barrier) filter which ensures none of the excitationlight source reaches the detector. Alternatively, these functions mayboth be accomplished by a single dichroic filter. The “fluorescencemicroscope” comprises any microscope that uses fluorescence to generatean image, whether it is a more simple set up like an epifluorescencemicroscope, or a more complicated design such as a confocal microscope,which uses optical sectioning to get better resolution of thefluorescent image.

In some embodiments, confocal microscopy is used for detection andimaging of the detection probe. Confocal microscopy uses pointillumination and a pinhole in an optically conjugate plane in front ofthe detector to eliminate out-of-focus signal. As only light produced byfluorescence very close to the focal plane can be detected, the image'soptical resolution, particularly in the sample depth direction, is muchbetter than that of wide-field microscopes. However, as much of thelight from sample fluorescence is blocked at the pinhole, this increasedresolution is at the cost of decreased signal intensity—so longexposures are often required. As only one point in the sample isilluminated at a time, 2D or 3D imaging requires scanning over a regularraster (e.g., a rectangular pattern of parallel scanning lines) in thespecimen. The achievable thickness of the focal plane is defined mostlyby the wavelength of the used light divided by the numerical aperture ofthe objective lens, but also by the optical properties of the specimen.The thin optical sectioning possible makes these types of microscopesparticularly good at 3D imaging and surface profiling of samples.CLARITY™-optimized light sheet microscopy (COLM) provides an alternativemicroscopy for fast 3D imaging of large clarified samples. COLMinterrogates large immunostained tissues, permits increased speed ofacquisition and results in a higher quality of generated data.

Other types of microscopy that can be employed comprise bright fieldmicroscopy, oblique illumination microscopy, dark field microscopy,phase contrast, differential interference contrast (DIC) microscopy,interference reflection microscopy (also known as reflected interferencecontrast, or RIC), single plane illumination microscopy (SPIM),super-resolution microscopy, laser microscopy, electron microscopy (EM),Transmission electron microscopy (TEM), Scanning electron microscopy(SEM), reflection electron microscopy (REM), Scanning transmissionelectron microscopy (STEM) and low-voltage electron microscopy (LVEM),scanning probe microscopy (SPM), atomic force microscopy (ATM),ballistic electron emission microscopy (BEEM), chemical force microscopy(CFM), conductive atomic force microscopy (C-AFM), electrochemicalscanning tunneling microscope (ECS™), electrostatic force microscopy(EFM), fluidic force microscope (FluidFM), force modulation microscopy(FMM), feature-oriented scanning probe microscopy (FOSPM), kelvin probeforce microscopy (KPFM), magnetic force microscopy (MFM), magneticresonance force microscopy (MRFM), near-field scanning opticalmicroscopy (NSOM) (or SNOM, scanning near-field optical microscopy,SNOM, Piezoresponse Force Microscopy (PFM), PS™, photon scanningtunneling microscopy (PS™), PTMS, photothermalmicrospectroscopy/microscopy (PTMS), SCM, scanning capacitancemicroscopy (SCM), SECM, scanning electrochemical microscopy (SECM), SGM,scanning gate microscopy (SGM), SHPM, scanning Hall probe microscopy(SHPM), SICM, scanning ion-conductance microscopy (SICM), SPSM spinpolarized scanning tunneling microscopy (SPSM), SSRM, scanning spreadingresistance microscopy (SSRM), SThM, scanning thermal microscopy (SThM),STM, scanning tunneling microscopy (STM), STP, scanning tunnelingpotentiometry (STP), SVM, scanning voltage microscopy (SVM), andsynchrotron x-ray scanning tunneling microscopy (SXS™), and intacttissue expansion microscopy (exM).

In some embodiments, sequencing can be performed in situ. In situsequencing typically involves incorporation of a labeled nucleotide(e.g., fluorescently labeled mononucleotides or dinucleotides) in asequential, template-dependent manner or hybridization of a labeledprimer (e.g., a labeled random hexamer) to a nucleic acid template suchthat the identities (e.g., nucleotide sequence) of the incorporatednucleotides or labeled primer extension products can be determined, andconsequently, the nucleotide sequence of the corresponding templatenucleic acid. Aspects of in situ sequencing are described, for example,in Mitra et al., (2003) Anal. Biochem. 320, 55-65, and Lee et al.,(2014) Science, 343(6177), 1360-1363. In addition, examples of methodsand systems for performing in situ sequencing are described in US2016/0024555, US 2019/0194709, and in U.S. Pat. Nos. 10,138,509,10,494,662 and 10,179,932. Exemplary techniques for in situ sequencingcomprise, but are not limited to, STARmap (described for example in Wanget al., (2018) Science, 361(6499) 5691), MERFISH (described for examplein Moffitt, (2016) Methods in Enzymology, 572, 1-49),hybridization-based in situ sequencing (HybISS) (described for examplein Gyllborg et al., Nucleic Acids Res (2020) 48(19):e112, and FISSEQ(described for example in US 2019/0032121). In some cases, sequencingcan be performed after the analytes are released from the biologicalsample.

In some embodiments, sequencing can be performed bysequencing-by-synthesis (SBS). In some embodiments, a sequencing primeris complementary to sequences at or near the one or more barcode(s). Insuch embodiments, sequencing-by-synthesis can comprise reversetranscription and/or amplification in order to generate a templatesequence from which a primer sequence can bind. Exemplary SBS methodscomprise those described for example, but not limited to, US2007/0166705, US 2006/0188901, U.S. Pat. No. 7,057,026, US 2006/0240439,US 2006/0281109, US 2011/005986, US 2005/0100900, U.S. Pat. No.9,217,178, US 2009/0118128, US 2012/0270305, US 2013/0260372, and US2013/0079232.

In some embodiments, sequence analysis of nucleic acids (e.g., nucleicacids such as RCA products comprising barcode sequences) can beperformed by sequential hybridization (e.g., sequencing by hybridizationand/or sequential in situ fluorescence hybridization). Sequentialfluorescence hybridization can involve sequential hybridization ofdetection probes comprising an oligonucleotide and a detectable label.In some embodiments, a method disclosed herein comprises sequentialhybridization of the detectable probes disclosed herein, includingdetectably labeled probes (e.g., fluorophore conjugatedoligonucleotides) and/or probes that are not detectably labeled per sebut are capable of binding (e.g., via nucleic acid hybridization) andbeing detected by detectably labeled probes. Exemplary methodscomprising sequential fluorescence hybridization of detectable probesare described in US 2019/0161796, US 2020/0224244, US 2022/0010358, US2021/0340618, and WO 2021/138676, all of which are incorporated hereinby reference.

In some embodiments, sequencing can be performed using single moleculesequencing by ligation. Such techniques utilize DNA ligase toincorporate oligonucleotides and identify the incorporation of sucholigonucleotides. The oligonucleotides typically have different labelsthat are correlated with the identity of a particular nucleotide in asequence to which the oligonucleotides hybridize. Aspects and featuresinvolved in sequencing by ligation are described, for example, inShendure et a1. Science (2005), 309: 1728-1732, and in U.S. Pat. Nos.5,599,675; 5,750,341; 6,969,488; 6,172,218; and 6,306,597.

In some embodiments, the barcodes of the probes (e.g., plurality ofprobes or probe sets) or complements or products thereof are targeted bydetectably labeled detection oligonucleotides, such as fluorescentlylabeled oligonucleotides. In some embodiments, one or more decodingschemes are used to decode the signals, such as fluorescence, forsequence determination. In any of the embodiments herein, barcodes(e.g., primary and/or secondary barcode sequences) can be analyzed(e.g., detected or sequenced) using any suitable methods or techniques,comprising those described herein, such as RNA sequential probing oftargets (RNA SPOTs), sequential fluorescent in situ hybridization(seqFISH), single-molecule fluorescent in situ hybridization (smFISH),multiplexed error-robust fluorescence in situ hybridization (MERFISH),hybridization-based in situ sequencing (HybISS), in situ sequencing,targeted in situ sequencing, fluorescent in situ sequencing (FISSEQ), orspatially-resolved transcript amplicon readout mapping (STARmap). Insome embodiments, the methods provided herein comprise analyzing thebarcodes by sequential hybridization and detection with a plurality oflabelled probes (e.g., detection oligonucleotides or detectable probes).Exemplary decoding schemes are described in Eng et al.,“Transcriptome-scale Super-Resolved Imaging in Tissues by RNA SeqFISH+,”Nature 568(7751):235-239 (2019); Chen et al., Science; 348(6233):aaa6090(2015); Gyllborg et al., Nucleic Acids Res (2020) 48(19):el 12; U.S.Pat. No. 10,457,980 B2; US 2016/0369329 A1; WO 2018/026873 A1; and US2017/0220733 A1, all of which are incorporated by reference in theirentirety. In some embodiments, these assays enable signal amplification,combinatorial decoding, and error correction schemes at the same time.

In some embodiments, nucleic acid hybridization can be used forsequencing. These methods utilize labeled nucleic acid decoder probesthat are complementary to at least a portion of a barcode sequence.Multiplex decoding can be performed with pools of many different probeswith distinguishable labels. Non-limiting examples of nucleic acidhybridization sequencing are described for example in U.S. Pat. No.8,460,865, and in Gunderson et al., Genome Research 14:870-877 (2004).

In some embodiments, real-time monitoring of DNA polymerase activity canbe used during sequencing. For example, nucleotide incorporations can bedetected through fluorescence resonance energy transfer (FRET), asdescribed for example in Levene et al., Science (2003), 299, 682-686,Lundquist et al., Opt. Lett. (2008), 33, 1026-1028, and Korlach et al.,Proc. Natl. Acad. Sci. USA (2008), 105, 1176-1181.

In some aspects, the analysis and/or sequence determination can becarried out at room temperature for best preservation of tissuemorphology with low background noise and error reduction. In someembodiments, the analysis and/or sequence determination compriseseliminating error accumulation as sequencing proceeds.

In some embodiments, the analysis and/or sequence determination involveswashing to remove unbound polynucleotides, thereafter revealing afluorescent product for imaging.

In some aspects, the provided embodiments can be applied to an in situmethod of analyzing target nucleic acid sequences (e.g., RNAs) and/orother targets (e.g., proteins) in intact tissues or samples in which thespatial information has been preserved. In some aspects, the embodimentscan be applied in an imaging or detection method for multiplexedanalysis of nucleic acids and/or other targets (e.g., proteins). In someaspects, the provided embodiments can be used to identify or detectregions and/or sequences of interest in target nucleic acids.

In some cases, analysis is performed on one or more images captured, andmay comprise processing the image(s) and/or quantifying signalsobserved. In some embodiments, a method disclosed herein comprisesmultiplexed analysis of a biological sample comprising consecutivecycles of probe hybridization, fluorescence imaging, and probe removal.In some embodiments, images of signals from different fluorescent and/ornon-fluorescent channels and/or detectable probe hybridization cyclescan be compared and analyzed. In some embodiments, images of signals (orabsence thereof) at a particular location in a sample from differentfluorescent channels and/or sequential detectable probe hybridizationcycles can be aligned to analyze an analyte at the location. Forinstance, a particular location in a sample can be tracked and signalspots from sequential hybridization cycles can be analyzed to detect atarget polynucleotide sequence (e.g., an associated barcode sequence orsubsequence thereof) at the location. The analysis may compriseprocessing information of one or more cell types, one or more types ofanalytes, a number or level of analyte, and/or a number or level ofcells detected in a particular region of the sample. In someembodiments, the analysis comprises detecting a sequence e.g., a barcodesequence present in an amplification product at a location in thesample. In some embodiments, the analysis includes quantification ofpuncta (e.g., if amplification products are detected). In some cases,the analysis includes determining whether particular cells and/orsignals are present that correlate with one or more analytes from aparticular panel. In some instances, the analysis includes using singlecell segmentation and resolution to determine cell type frequencies in aregion of interest of a sample. In some embodiments, the obtainedinformation may be compared to a positive and negative control, toanother selected region of interest, or to a threshold of a feature todetermine if the region of interest exhibits a certain feature orphenotype. In some cases, the information may comprise signals from acell, a region, and/or comprise readouts from multiple detectablelabels. In some case, the analysis further includes displaying theinformation from the analysis or detection step. In some embodiments,software may be used to automate the processing, analysis, and/ordisplay of data.

IV. Samples and Sample Processing

A sample disclosed herein can be or derived from any biological sample.Methods, probes, and kits disclosed herein may be used for analyzing abiological sample, which may be obtained from a subject using any of avariety of techniques including, but not limited to, biopsy, surgery,and laser capture microscopy (LCM), and generally comprises cells and/orother biological material from the subject. In addition to the subjectsdescribed above, a biological sample can be obtained from a prokaryotesuch as a bacterium, an archaea, a virus, or a viroid. A biologicalsample can also be obtained from non-mammalian organisms (e.g., a plant,an insect, an arachnid, a nematode, a fungus, or an amphibian). Abiological sample can also be obtained from a eukaryote, such as atissue sample, a patient derived organoid (PDO) or patient derivedxenograft (PDX). A biological sample from an organism may comprise oneor more other organisms or components therefrom. For example, amammalian tissue section may comprise a prion, a viroid, a virus, abacterium, a fungus, or components from other organisms, in addition tomammalian cells and non-cellular tissue components. Subjects from whichbiological samples can be obtained can be healthy or asymptomaticindividuals, individuals that have or are suspected of having a disease(e.g., a patient with a disease such as cancer) or a pre-disposition toa disease, and/or individuals in need of therapy or suspected of needingtherapy.

The biological sample can include any number of macromolecules, forexample, cellular macromolecules and organelles (e.g., mitochondria andnuclei). The biological sample can be obtained as a tissue sample, suchas a tissue section, biopsy, a core biopsy, needle aspirate, or fineneedle aspirate. The sample can be a fluid sample, such as a bloodsample, urine sample, or saliva sample. The sample can be a skin sample,a colon sample, a cheek swab, a histology sample, a histopathologysample, a plasma or serum sample, a tumor sample, living cells, culturedcells, a clinical sample such as, for example, whole blood orblood-derived products, blood cells, or cultured tissues or cells,including cell suspensions. In some embodiments, the biological samplemay comprise cells which are deposited on a surface.

Biological samples can be derived from a homogeneous culture orpopulation of the subjects or organisms mentioned herein oralternatively from a collection of several different organisms, forexample, in a community or ecosystem.

Biological samples can include one or more diseased cells. A diseasedcell can have altered metabolic properties, gene expression, proteinexpression, and/or morphologic features. Examples of diseases includeinflammatory disorders, metabolic disorders, nervous system disorders,and cancer. Cancer cells can be derived from solid tumors, hematologicalmalignancies, cell lines, or obtained as circulating tumor cells.Biological samples can also include fetal cells and immune cells.

In some embodiments, the sample is a cell pellet or cell block. In someembodiments, the biological sample comprises a tissue sample. In someinstances, the tissue sample is a tissue biopsy. In some instances, thebiological sample is a tumor biopsy. In some instances, the biologicalsample is a surgical resection. In some instances, the biological samplecomprises a tumor or a portion of a tumor.

Biological samples can include analytes (e.g., protein, RNA, and/or DNA)embedded in a 3D matrix. In some embodiments, amplicons (e.g., rollingcircle amplification products) derived from or associated with analytes(e.g., protein, RNA, and/or DNA) can be embedded in a 3D matrix. In someembodiments, a 3D matrix may comprise a network of natural moleculesand/or synthetic molecules that are chemically and/or enzymaticallylinked, e.g., by crosslinking. In some embodiments, a 3D matrix maycomprise a synthetic polymer. In some embodiments, a 3D matrix comprisesa hydrogel.

In some embodiments, a substrate herein can be any support that isinsoluble in aqueous liquid and which allows for positioning ofbiological samples, analytes, features, and/or reagents on the support.In some embodiments, a biological sample can be attached to a substrate.Attachment of the biological sample can be irreversible or reversible,depending upon the nature of the sample and subsequent steps in theanalytical method. In certain embodiments, the sample can be attached tothe substrate reversibly by applying a suitable polymer coating to thesubstrate, and contacting the sample to the polymer coating. The samplecan then be detached from the substrate, e.g., using an organic solventthat at least partially dissolves the polymer coating. Hydrogels areexamples of polymers that are suitable for this purpose.

In some embodiments, the substrate can be coated or functionalized withone or more substances to facilitate attachment of the sample to thesubstrate. Suitable substances that can be used to coat or functionalizethe substrate include, but are not limited to, lectins, poly-lysine,antibodies, and polysaccharides.

A variety of steps can be performed to prepare or process a biologicalsample for and/or during an assay. Except where indicated otherwise, thepreparative or processing steps described below can generally becombined in any manner and in any order to appropriately prepare orprocess a particular sample for and/or analysis.

(i) Tissue Sectioning

A biological sample can be harvested from a subject (e.g., via surgicalbiopsy, whole subject sectioning) or grown in vitro on a growthsubstrate or culture dish as a population of cells, and prepared foranalysis as a tissue slice or tissue section. Grown samples may besufficiently thin for analysis without further processing steps.Alternatively, grown samples, and samples obtained via biopsy orsectioning, can be prepared as thin tissue sections using a mechanicalcutting apparatus such as a vibrating blade microtome. As anotheralternative, in some embodiments, a thin tissue section can be preparedby applying a touch imprint of a biological sample to a suitablesubstrate material.

The thickness of the tissue section can be a fraction of (e.g., lessthan 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1) the maximumcross-sectional dimension of a cell. However, tissue sections having athickness that is larger than the maximum cross-section cell dimensioncan also be used. For example, cryostat sections can be used, which canbe, e.g., 10-20 μm thick.

More generally, the thickness of a tissue section typically depends onthe method used to prepare the section and the physical characteristicsof the tissue, and therefore sections having a wide variety of differentthicknesses can be prepared and used. For example, the thickness of thetissue section can be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 20, 30, 40, or 50 μm.Thicker sections can also be used if desired or convenient, e.g., atleast 70, 80, 90, or 100 μm or more. Typically, the thickness of atissue section is between 1-100 μm, 1-50 μm, 1-30 μm, 1-25 μm, 1-20 μm,1-15 μm, 1-10 μm, 2-8 μm, 3-7 μm, or 4-6 μm, but as mentioned above,sections with thicknesses larger or smaller than these ranges can alsobe analysed.

Multiple sections can also be obtained from a single biological sample.For example, multiple tissue sections can be obtained from a surgicalbiopsy sample by performing serial sectioning of the biopsy sample usinga sectioning blade. Spatial information among the serial sections can bepreserved in this manner, and the sections can be analysed successivelyto obtain three-dimensional information about the biological sample.

(ii) Freezing

In some embodiments, the biological sample (e.g., a tissue section asdescribed above) can be prepared by deep freezing at a temperaturesuitable to maintain or preserve the integrity (e.g., the physicalcharacteristics) of the tissue structure. The frozen tissue sample canbe sectioned, e.g., thinly sliced, onto a substrate surface using anynumber of suitable methods. For example, a tissue sample can be preparedusing a chilled microtome (e.g., a cryostat) set at a temperaturesuitable to maintain both the structural integrity of the tissue sampleand the chemical properties of the nucleic acids in the sample. Such atemperature can be, e.g., less than −15° C., less than −20° C., or lessthan −25° C.

(iii) Fixation and Postfixation

In some embodiments, the biological sample can be prepared usingformalin-fixation and paraffin-embedding (FFPE), which are establishedmethods. In some embodiments, cell suspensions and other non-tissuesamples can be prepared using formalin-fixation and paraffin-embedding.Following fixation of the sample and embedding in a paraffin or resinblock, the sample can be sectioned as described above. Prior toanalysis, the paraffin-embedding material can be removed from the tissuesection (e.g., deparaffinization) by incubating the tissue section in anappropriate solvent (e.g., xylene) followed by a rinse (e.g., 99.5%ethanol for 2 minutes, 96% ethanol for 2 minutes, and 70% ethanol for 2minutes).

As an alternative to formalin fixation described above, a biologicalsample can be fixed in any of a variety of other fixatives to preservethe biological structure of the sample prior to analysis. For example, asample can be fixed via immersion in ethanol, methanol, acetone,paraformaldehyde (PFA)-Triton, and combinations thereof.

In some embodiments, acetone fixation is used with fresh frozen samples,which can include, but are not limited to, cortex tissue, mouseolfactory bulb, human brain tumor, human post-mortem brain, and breastcancer samples. When acetone fixation is performed, pre-permeabilizationsteps (described below) may not be performed. Alternatively, acetonefixation can be performed in conjunction with permeabilization steps.

In some embodiments, the methods provided herein comprises one or morepost-fixing (also referred to as postfixation) steps. In someembodiments, one or more post-fixing step is performed after contactinga sample with a polynucleotide disclosed herein, e.g., one or moreprobes such as a circular or circularizable probe (e.g., a padlockprobe). In some embodiments, one or more post-fixing step is performedafter a hybridization complex comprising a probe and a target is formedin a sample. In some embodiments, one or more post-fixing step isperformed prior to a ligation reaction disclosed herein, such as theligation to circularize a circularizable probe or probe set.

In some embodiments, one or more post-fixing step is performed aftercontacting a sample with a binding or labelling agent (e.g., an antibodyor antigen binding fragment thereof) for a non-nucleic acid analyte suchas a protein analyte. The labelling agent can comprise a nucleic acidmolecule (e.g., reporter oligonucleotide) comprising a sequencecorresponding to the labelling agent and therefore corresponds to (e.g.,uniquely identifies) the analyte. In some embodiments, the labellingagent can comprise a reporter oligonucleotide comprising one or morebarcode sequences.

A post-fixing step may be performed using any suitable fixation reagentdisclosed herein, for example, 3% (w/v) paraformaldehyde in DEPC-PBS.

(iv) Embedding

As an alternative to paraffin embedding described above, a biologicalsample can be embedded in any of a variety of other embedding materialsto provide structural substrate to the sample prior to sectioning andother handling steps. In some cases, the embedding material can beremoved e.g., prior to analysis of tissue sections obtained from thesample. Suitable embedding materials include, but are not limited to,waxes, resins (e.g., methacrylate resins), epoxies, and agar.

In some embodiments, the biological sample can be embedded in a matrix(e.g., a hydrogel matrix). Embedding the sample in this manner typicallyinvolves contacting the biological sample with a hydrogel such that thebiological sample becomes surrounded by the hydrogel. For example, thesample can be embedded by contacting the sample with a suitable polymermaterial, and activating the polymer material to form a hydrogel. Insome embodiments, the hydrogel is formed such that the hydrogel isinternalized within the biological sample.

In some embodiments, the biological sample is immobilized in thehydrogel via cross-linking of the polymer material that forms thehydrogel. Cross-linking can be performed chemically and/orphotochemically, or alternatively by any other hydrogel-formationmethod.

The composition and application of the hydrogel-matrix to a biologicalsample typically depends on the nature and preparation of the biologicalsample (e.g., sectioned, non-sectioned, type of fixation). As oneexample, where the biological sample is a tissue section, thehydrogel-matrix can include a monomer solution and an ammoniumpersulfate (APS) initiator/tetramethylethylenediamine (TEMED)accelerator solution. As another example, where the biological sampleconsists of cells (e.g., cultured cells or cells disassociated from atissue sample), the cells can be incubated with the monomer solution andAPS/TEMED solutions. For cells, hydrogel-matrix gels are formed incompartments, including but not limited to devices used to culture,maintain, or transport the cells. For example, hydrogel-matrices can beformed with monomer solution plus APS/TEMED added to the compartment toa depth ranging from about 0.1 m to about 2 mm.

Additional methods and aspects of hydrogel embedding of biologicalsamples are described for example in Chen et al., Science347(6221):543-548, 2015, the entire contents of which are incorporatedherein by reference.

(v) Staining and Immunohistochemistry (IHC)

To facilitate visualization, biological samples can be stained using awide variety of stains and staining techniques. In some embodiments, forexample, a sample can be stained using any number of stains and/orimmunohistochemical reagents. One or more staining steps may beperformed to prepare or process a biological sample for an assaydescribed herein or may be performed during and/or after an assay. Insome embodiments, the sample can be contacted with one or more nucleicacid stains, membrane stains (e.g., cellular or nuclear membrane),cytological stains, or combinations thereof. In some examples, the stainmay be specific to proteins, phospholipids, DNA (e.g., dsDNA, ssDNA),RNA, an organelle or compartment of the cell. The sample may becontacted with one or more labeled antibodies (e.g., a primary antibodyspecific for the analyte of interest and a labeled secondary antibodyspecific for the primary antibody). In some embodiments, cells in thesample can be segmented using one or more images taken of the stainedsample.

In some embodiments, the stain is performed using a lipophilic dye. Insome examples, the staining is performed with a lipophilic carbocyanineor aminostyryl dye, or analogs thereof (e.g., DiI, DiO, DiR, DiD). Othercell membrane stains may include FM and RH dyes or immunohistochemicalreagents specific for cell membrane proteins. In some examples, thestain may include but is not limited to, acridine orange, acid fuchsin,Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin,ethidium bromide, acid fuchsine, haematoxylin, Hoechst stains, iodine,methyl green, methylene blue, neutral red, Nile blue, Nile red, osmiumtetroxide, ruthenium red, propidium iodide, rhodamine (e.g., rhodamineB), or safranine, or derivatives thereof. In some embodiments, thesample may be stained with haematoxylin and eosin (H&E).

The sample can be stained using hematoxylin and eosin (H&E) stainingtechniques, using Papanicolaou staining techniques, Masson's trichromestaining techniques, silver staining techniques, Sudan stainingtechniques, and/or using Periodic Acid Schiff (PAS) staining techniques.PAS staining is typically performed after formalin or acetone fixation.In some embodiments, the sample can be stained using Romanowsky stain,including Wright's stain, Jenner's stain, Can-Grunwald stain, Leishmanstain, and Giemsa stain.

In some embodiments, biological samples can be destained. Methods ofdestaining or discoloring a biological sample generally depend on thenature of the stain(s) applied to the sample. For example, in someembodiments, one or more immunofluorescent stains are applied to thesample via antibody coupling. Such stains can be removed usingtechniques such as cleavage of disulfide linkages via treatment with areducing agent and detergent washing, chaotropic salt treatment,treatment with antigen retrieval solution, and treatment with an acidicglycine buffer. Methods for multiplexed staining and destaining aredescribed, for example, in Bolognesi et al., J. Histochem. Cytochem.2017; 65(8): 431-444, Lin et al., Nat Commun. 2015; 6:8390, Pirici etal., J. Histochem. Cytochem. 2009; 57:567-75, and Glass et al., J.Histochem. Cytochem. 2009; 57:899-905, the entire contents of each ofwhich are incorporated herein by reference.

(vi) Isometric Expansion

In some embodiments, a biological sample embedded in a matrix (e.g., ahydrogel) can be isometrically expanded. Isometric expansion methodsthat can be used include hydration, a preparative step in expansionmicroscopy, as described in Chen et al., Science 347(6221):543-548,2015.

Isometric expansion can be performed by anchoring one or more componentsof a biological sample to a gel, followed by gel formation, proteolysis,and swelling. In some embodiments, analytes in the sample, products ofthe analytes, and/or probes associated with analytes in the sample canbe anchored to the matrix (e.g., hydrogel). Isometric expansion of thebiological sample can occur prior to immobilization of the biologicalsample on a substrate, or after the biological sample is immobilized toa substrate. In some embodiments, the isometrically expanded biologicalsample can be removed from the substrate prior to contacting thesubstrate with probes disclosed herein.

In general, the steps used to perform isometric expansion of thebiological sample can depend on the characteristics of the sample (e.g.,thickness of tissue section, fixation, cross-linking), and/or theanalyte of interest (e.g., different conditions to anchor RNA, DNA, andprotein to a gel).

In some embodiments, proteins in the biological sample are anchored to aswellable gel such as a polyelectrolyte gel. An antibody can be directedto the protein before, after, or in conjunction with being anchored tothe swellable gel. DNA and/or RNA in a biological sample can also beanchored to the swellable gel via a suitable linker. Examples of suchlinkers include, but are not limited to, 6-((Acryloyl)amino) hexanoicacid (Acryloyl-X SE) (available from ThermoFisher, Waltham, MA),Label-IT Amine (available from MirusBio, Madison, WI) and Label X(described for example in Chen et al., Nat. Methods 13:679-684, 2016,the entire contents of which are incorporated herein by reference).

Isometric expansion of the sample can increase the spatial resolution ofthe subsequent analysis of the sample. The increased resolution inspatial profiling can be determined by comparison of an isometricallyexpanded sample with a sample that has not been isometrically expanded.

In some embodiments, a biological sample is isometrically expanded to asize at least 2×, 2.1×, 2.2×, 2.3×, 2.4×, 2.5×, 2.6×, 2.7×, 2.8×, 2.9×,3×, 3.1×, 3.2×, 3.3×, 3.4×, 3.5×, 3.6×, 3.7×, 3.8×, 3.9×, 4×, 4.1×,4.2×, 4.3×, 4.4×, 4.5×, 4.6×, 4.7×, 4.8×, or 4.9× its non-expanded size.In some embodiments, the sample is isometrically expanded to at least 2×and less than 20× of its non-expanded size.

(vii) Crosslinking and De-Crosslinking

In some embodiments, the biological sample is reversibly cross-linkedprior to or during an in situ assay. In some aspects, the analytes,polynucleotides and/or amplification product (e.g., amplicon) of ananalyte or a probe bound thereto can be anchored to a polymer matrix.For example, the polymer matrix can be a hydrogel. In some embodiments,one or more of the polynucleotide probe(s) and/or amplification product(e.g., amplicon) thereof can be modified to contain functional groupsthat can be used as an anchoring site to attach the polynucleotideprobes and/or amplification product to a polymer matrix. In someembodiments, a modified probe comprising oligo dT may be used to bind tomRNA molecules of interest, followed by reversible crosslinking of themRNA molecules.

In some embodiments, the biological sample is immobilized in a hydrogelvia cross-linking of the polymer material that forms the hydrogel.Cross-linking can be performed chemically and/or photochemically, oralternatively by any other hydrogel-formation method. A hydrogel mayinclude a macromolecular polymer gel including a network. Within thenetwork, some polymer chains can optionally be cross-linked, althoughcross-linking does not always occur.

In some embodiments, a hydrogel can include hydrogel subunits, such as,but not limited to, acrylamide, bis-acrylamide, polyacrylamide andderivatives thereof, poly(ethylene glycol) and derivatives thereof (e.g.PEG-acrylate (PEG-DA), PEG-RGD), gelatin-methacryloyl (GelMA),methacrylated hyaluronic acid (MeHA), polyaliphatic polyurethanes,polyether polyurethanes, polyester polyurethanes, polyethylenecopolymers, polyamides, polyvinyl alcohols, polypropylene glycol,polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide,poly(hydroxyethyl acrylate), and poly(hydroxyethyl methacrylate),collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin,alginate, protein polymers, methylcellulose, and the like, andcombinations thereof.

In some embodiments, a hydrogel comprises a hybrid material, e.g., thehydrogel material comprises elements of both synthetic and naturalpolymers. Examples of suitable hydrogels are described, for example, inU.S. Pat. Nos. 6,391,937, 9,512,422, and 9,889,422, and in U.S. PatentApplication Publication Nos. 2017/0253918, 2018/0052081 and2010/0055733, the entire contents of each of which are incorporatedherein by reference.

In some embodiments, the hydrogel can form the substrate. In someembodiments, the substrate comprises a hydrogel and one or more secondmaterials. In some embodiments, the hydrogel is placed on top of one ormore second materials. For example, the hydrogel can be pre-formed andthen placed on top of, underneath, or in any other configuration withone or more second materials. In some embodiments, hydrogel formationoccurs after contacting one or more second materials during formation ofthe substrate. Hydrogel formation can also occur within a structure(e.g., wells, ridges, projections, and/or markings) located on asubstrate.

In some embodiments, hydrogel formation on a substrate occurs before,contemporaneously with, or after probes are provided to the sample. Forexample, hydrogel formation can be performed on the substrate alreadycontaining the probes.

In some embodiments, hydrogel formation occurs within a biologicalsample. In some embodiments, a biological sample (e.g., tissue section)is embedded in a hydrogel. In some embodiments, hydrogel subunits areinfused into the biological sample, and polymerization of the hydrogelis initiated by an external or internal stimulus.

In embodiments in which a hydrogel is formed within a biological sample,functionalization chemistry can be used. In some embodiments,functionalization chemistry includes hydrogel-tissue chemistry (HTC).Any hydrogel-tissue backbone (e.g., synthetic or native) suitable forHTC can be used for anchoring biological macromolecules and modulatingfunctionalization. Non-limiting examples of methods using HTC backbonevariants include CLARITY, PACT, ExM, SWITCH and ePACT. In someembodiments, hydrogel formation within a biological sample is permanent.For example, biological macromolecules can permanently adhere to thehydrogel allowing multiple rounds of interrogation. In some embodiments,hydrogel formation within a biological sample is reversible.

In some embodiments, additional reagents are added to the hydrogelsubunits before, contemporaneously with, and/or after polymerization.For example, additional reagents can include but are not limited tooligonucleotides (e.g., probes), endonucleases to fragment DNA,fragmentation buffer for DNA, DNA polymerase enzymes, dNTPs used toamplify the nucleic acid and to attach the barcode to the amplifiedfragments. Other enzymes can be used, including without limitation, RNApolymerase, ligase, proteinase K, and DNAse. Additional reagents canalso include reverse transcriptase enzymes, including enzymes withterminal transferase activity, primers, and switch oligonucleotides. Insome embodiments, optical labels are added to the hydrogel subunitsbefore, contemporaneously with, and/or after polymerization.

In some embodiments, HTC reagents are added to the hydrogel before,contemporaneously with, and/or after polymerization. In someembodiments, a cell labelling agent is added to the hydrogel before,contemporaneously with, and/or after polymerization. In someembodiments, a cell-penetrating agent is added to the hydrogel before,contemporaneously with, and/or after polymerization.

Hydrogels embedded within biological samples can be cleared using anysuitable method. For example, electrophoretic tissue clearing methodscan be used to remove biological macromolecules from thehydrogel-embedded sample. In some embodiments, a hydrogel-embeddedsample is stored before or after clearing of hydrogel, in a medium(e.g., a mounting medium, methylcellulose, or other semi-solid mediums).

In some embodiments, a method disclosed herein comprises de-crosslinkingthe reversibly cross-linked biological sample. The de-crosslinking doesnot need to be complete. In some embodiments, only a portion ofmolecular crosslinks in the reversibly cross-linked biological sampleare de-crosslinked.

(viii) Tissue Permeabilization and Treatment

In some embodiments, a biological sample can be permeabilized tofacilitate transfer of species (such as probes) into the sample. If asample is not permeabilized sufficiently, probes that enter the sampleand bind to analytes therein may be too low to enable adequate analysis.Conversely, if the tissue sample is too permeable, the relative spatialrelationship of the analytes within the tissue sample can be lost.Hence, a balance between permeabilizing the tissue sample enough toobtain good signal intensity while still maintaining the spatialresolution of the analyte distribution in the sample is desirable.

In general, a biological sample can be permeabilized by exposing thesample to one or more permeabilizing agents. Suitable agents for thispurpose include, but are not limited to, organic solvents (e.g.,acetone, ethanol, and methanol), cross-linking agents (e.g.,paraformaldehyde), detergents (e.g., saponin, Triton X-100™ orTween-20™), and enzymes (e.g., trypsin, proteases). In some embodiments,the biological sample can be incubated with a cellular permeabilizingagent to facilitate permeabilization of the sample. Additional methodsfor sample permeabilization are described, for example, in Jamur et al.,Method Mol. Biol. 588:63-66, 2010, the entire contents of which areincorporated herein by reference. Any suitable method for samplepermeabilization can generally be used in connection with the samplesdescribed herein.

In some embodiments, the biological sample can be permeabilized byadding one or more lysis reagents to the sample. Examples of suitablelysis agents include, but are not limited to, bioactive reagents such aslysis enzymes that are used for lysis of different cell types, e.g.,gram positive or negative bacteria, plants, yeast, mammalian, such aslysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase,and a variety of other commercially available lysis enzymes.

Other lysis agents can additionally or alternatively be added to thebiological sample to facilitate permeabilization. For example,surfactant-based lysis solutions can be used to lyse sample cells. Lysissolutions can include ionic surfactants such as, for example, sarcosyland sodium dodecyl sulfate (SDS). More generally, chemical lysis agentscan include, without limitation, organic solvents, chelating agents,detergents, surfactants, and chaotropic agents.

In some embodiments, the biological sample can be permeabilized bynon-chemical permeabilization methods. Non-chemical permeabilizationmethods that can be used herein include, but are not limited to,physical lysis techniques such as electroporation, mechanicalpermeabilization methods (e.g., bead beating using a homogenizer andgrinding balls to mechanically disrupt sample tissue structures),acoustic permeabilization (e.g., sonication), and thermal lysistechniques such as heating to induce thermal permeabilization of thesample.

Additional reagents can be added to a biological sample to performvarious functions prior to analysis of the sample. In some embodiments,DNase and RNase inactivating agents or inhibitors such as proteinase K,and/or chelating agents such as EDTA, can be added to the sample. Forexample, a method disclosed herein may comprise a step for increasingaccessibility of a nucleic acid for binding, e.g., a denaturation stepto open up DNA in a cell for hybridization by a probe. For example,proteinase K treatment may be used to free up DNA with proteins boundthereto.

(ix) Selective Enrichment of RNA Species

In some embodiments, where RNA is the analyte, one or more RNA analytespecies of interest can be selectively enriched. For example, one ormore species of RNA of interest can be selected by addition of one ormore oligonucleotides to the sample. In some embodiments, the additionaloligonucleotide is a sequence used for priming a reaction by an enzyme(e.g., a polymerase). For example, one or more primer sequences withsequence complementarity to one or more RNAs of interest can be used toamplify the one or more RNAs of interest, thereby selectively enrichingthese RNAs.

In some aspects, when two or more analytes are analyzed, a first andsecond probe that is specific for (e.g., specifically hybridizes to)each RNA or cDNA analyte are used. For example, in some embodiments ofthe methods provided herein, templated ligation is used to detect geneexpression in a biological sample. An analyte of interest (such as aprotein), bound by a labelling agent or binding agent (e.g., an antibodyor epitope binding fragment thereof), wherein the binding agent isconjugated or otherwise associated with a reporter oligonucleotidecomprising a reporter sequence that identifies the binding agent, can betargeted for analysis. Probes may be hybridized to the reporteroligonucleotide and ligated in a templated ligation reaction to generatea product for analysis. In some embodiments, gaps between the probeoligonucleotides may first be filled prior to ligation, using, forexample, Mu polymerase, DNA polymerase, RNA polymerase, reversetranscriptase, VENT polymerase, Taq polymerase, and/or any combinations,derivatives, and variants (e.g., engineered mutants) thereof. In someembodiments, the assay can further include amplification of templatedligation products (e.g., by multiplex PCR).

Alternatively, one or more species of RNA can be down-selected (e.g.,removed) using any of a variety of methods. For example, probes can beadministered to a sample that selectively hybridize to ribosomal RNA(rRNA), thereby reducing the pool and concentration of rRNA in thesample. Additionally and alternatively, duplex-specific nuclease (DSN)treatment can remove rRNA (see, e.g., Archer, et a1, Selective andflexible depletion of problematic sequences from RNA-seq libraries atthe cDNA stage, BMC Genomics, 15 401, (2014), the entire contents ofwhich are incorporated herein by reference). Furthermore, hydroxyapatitechromatography can remove abundant species (e.g., rRNA) (see, e.g.,Vandernoot, V.A., cDNA normalization by hydroxyapatite chromatography toenrich transcriptome diversity in RNA-seq applications, Biotechniques,53(6) 373-80, (2012), the entire contents of which are incorporatedherein by reference).

V. Compositions and Kits

In some embodiments, provided herein are kits, for example comprising acompound disclosed herein (e.g., in Section II-C) for catalyticde-crosslinking of a fixed biological sample. In some embodiments, thecompound is provided in a composition (e.g., a composition comprisingDMSO) and the kit can further comprise one or more other compositions,e.g., a buffer for the compound. In some examples, a solution or asuspension comprising the catalyst and a buffer is provided. In someinstances, the buffer comprises citrate, tris(hydroxymethyl)aminomethane(Tris) phosphate-buffered saline (PBS),2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES),2-(N-morpholino)ethanesulfonic acid (MES), or a combination thereof. Insome instances, the solution or suspension comprises sodium dodecylsulfate (SDS), urea, and/or a proteinase (e.g., proteinase K). The kitscan comprise one or more reagents required for one or more stepscomprising hybridization, ligation, extension, detection, and/or samplepreparation as described herein. In some embodiments, the kit comprisesone or more labelling agents, e.g., disclosed in Section III-B andSection III-C. In some embodiments, the kit comprises one or moreoligonucleotides, e.g., nucleic acid probes disclosed in Section III-Band Section III-C, for detecting one or more nucleic acid analytesand/or one or more non-nucleic acid analytes. In some embodiments, thekit comprises one or more antibodies (e.g., for detecting proteinanalytes) which can be optionally labelled with a detectable label suchas a fluorophore and/or a reporter oligonucleotide.

The various components of the kit may be present in separate containersor certain compatible components may be pre-combined into a singlecontainer. In some embodiments, the kits further contain instructionsfor using the components of the kit to practice the provided methods.

In some embodiments, the kits can contain reagents and/or consumablesrequired for performing one or more steps of the provided methods. Insome embodiments, the kits contain reagents for fixing (e.g.,crosslinking), embedding, and/or permeabilizing the biological sample.In some embodiments, the kits contain reagents, such as enzymes andbuffers. In some aspects, the kit can also comprise any of the reagentsdescribed herein, e.g., wash buffer. In some embodiments, the kitscontain reagents for detection and/or sequencing, such as barcodedetection probes or detectable labels. In some embodiments, the kitsoptionally contain other components, for example nucleic acid primers,enzymes and reagents, buffers, nucleotides, and reagents for additionalassays.

VI. Applications

In some aspects, the provided embodiments can be applied in an in situmethod of analyzing nucleic acid sequences, such as fluorescent in situhybridization (FISH)-based methods, in situ transcriptomic analysis orin situ sequencing, for example from intact tissues or samples in whichthe spatial information has been preserved. In some aspects, theembodiments can be applied in an imaging or detection method formultiplexed nucleic acid analysis. In some aspects, the providedembodiments can be used to detect a signal associated with a detectablelabel of a nucleic acid probe that is hybridized to a target sequence ofa target nucleic acid in a biological sample.

In some embodiments, the target nucleic acid comprises asingle-nucleotide polymorphism (SNP). In some embodiments, the targetnucleic acid comprises is a single-nucleotide variant (SNV). In someembodiments, the target nucleic acid comprises a single-nucleotidesubstitution. In some embodiments, the target nucleic acid comprises apoint mutation. In some embodiments, the target nucleic acid comprises asingle-nucleotide insertion.

In some aspects, the embodiments can be applied in investigative and/ordiagnostic applications, for example, for characterization or assessmentof particular cell or a tissue from a subject. Applications of theprovided method can comprise biomedical research and clinicaldiagnostics. For example, in biomedical research, applications comprise,but are not limited to, spatially resolved gene expression analysis forbiological investigation or drug screening. In clinical diagnostics,applications comprise, but are not limited to, detecting gene markerssuch as disease, immune responses, bacterial or viral DNA/RNA forpatient samples.

In some aspects, the embodiments can be applied to visualize thedistribution of genetically encoded markers in whole tissue atsubcellular resolution, for example, chromosomal abnormalities(inversions, duplications, translocations, etc.), loss of geneticheterozygosity, the presence of gene alleles indicative of apredisposition towards disease or good health, likelihood ofresponsiveness to therapy, or in personalized medicine or ancestry.

VII. Terminology

Unless defined otherwise, all terms of art, notations and othertechnical and scientific terms or terminology used herein are intendedto have the same meaning as is commonly understood by one of ordinaryskill in the art to which the claimed subject matter pertains. In somecases, terms with commonly understood meanings are defined herein forclarity and/or for ready reference, and the inclusion of suchdefinitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art.

The terms “polynucleotide,” “polynucleotide,” and “nucleic acidmolecule”, used interchangeably herein, refer to polymeric forms ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. Thus, this term comprises, but is not limited to,single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA,DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases orother natural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases. The backbone of the polynucleotide cancomprise sugars and phosphate groups (as may typically be found in RNAor DNA), or modified or substituted sugar or phosphate groups.

“Hybridization” as used herein may refer to the process in which twosingle-stranded polynucleotides bind non-covalently to form a stabledouble-stranded polynucleotide. In one aspect, the resultingdouble-stranded polynucleotide can be a “hybrid” or “duplex.”“Hybridization conditions” typically include salt concentrations ofapproximately less than 1 M, often less than about 500 mM and may beless than about 200 mM. A “hybridization buffer” includes a bufferedsalt solution such as 5% SSPE, or other such buffers known in the art.Hybridization temperatures can be as low as 5° C., but are typicallygreater than 22° C., and more typically greater than about 30° C., andtypically in excess of 37° C. Hybridizations are often performed understringent conditions, e.g., conditions under which a sequence willhybridize to its target sequence but will not hybridize to other,non-complementary sequences. Stringent conditions are sequence-dependentand are different in different circumstances. For example, longerfragments may require higher hybridization temperatures for specifichybridization than short fragments. As other factors may affect thestringency of hybridization, including base composition and length ofthe complementary strands, presence of organic solvents, and the extentof base mismatching, the combination of parameters is more importantthan the absolute measure of any one parameter alone. Generallystringent conditions are selected to be about 5° C. lower than the T_(m)for the specific sequence at a defined ionic strength and pH. Themelting temperature T_(m) can be the temperature at which a populationof double-stranded nucleic acid molecules becomes half dissociated intosingle strands. Several equations for calculating the T_(m) of nucleicacids are well known in the art. As indicated by standard references, asimple estimate of the T_(m) value may be calculated by the equation,T_(m)=81.5+0.41 (% G+C), when a nucleic acid is in aqueous solution at 1M NaCl (see e.g., Anderson and Young, Quantitative Filter Hybridization,in Nucleic Acid Hybridization (1985)). Other references (e.g., Allawiand SantaLucia, Jr., Biochemistry, 36:10581-94 (1997)) includealternative methods of computation which take structural andenvironmental, as well as sequence characteristics into account for thecalculation of T_(m).

In general, the stability of a hybrid is a function of the ionconcentration and temperature. Typically, a hybridization reaction isperformed under conditions of lower stringency, followed by washes ofvarying, but higher, stringency. Exemplary stringent conditions includea salt concentration of at least 0.01 M to no more than 1 M sodium ionconcentration (or other salt) at a pH of about 7.0 to about 8.3 and atemperature of at least 25° C. For example, conditions of 5×SSPE (750 mMNaCl, 50 mM sodium phosphate, 5 mM EDTA at pH 7.4) and a temperature ofapproximately 30° C. are suitable for allele-specific hybridizations,though a suitable temperature depends on the length and/or GC content ofthe region hybridized. In one aspect, “stringency of hybridization” indetermining percentage mismatch can be as follows: 1) high stringency:0.1×SSPE, 0.1% SDS, 65° C.; 2) medium stringency: 0.2×SSPE, 0.1% SDS,50° C. (also referred to as moderate stringency); and 3) low stringency:1.0×SSPE, 0.1% SDS, 50° C. It is understood that equivalent stringenciesmay be achieved using alternative buffers, salts and temperatures. Forexample, moderately stringent hybridization can refer to conditions thatpermit a nucleic acid molecule such as a probe to bind a complementarynucleic acid molecule. The hybridized nucleic acid molecules generallyhave at least 60% identity, including for example at least any of 70%,75%, 80%, 85%, 90%, or 95% identity. Moderately stringent conditions canbe conditions equivalent to hybridization in 50% formamide, 5×Denhardt'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. High stringency conditions can be provided, forexample, by hybridization in 50% formamide, 5×Denhardt's solution,5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE, and 0.1%SDS at 65° C. Low stringency hybridization can refer to conditionsequivalent to hybridization in 10% formamide, 5×Denhardt's solution,6×SSPE, 0.2% SDS at 22° C., followed by washing in 1×SSPE, 0.2% SDS, at37° C. Denhardt's solution contains 1% Ficoll, 1% polyvinylpyrolidone,and 1% bovine serum albumin (BSA). 20×SSPE (sodium chloride, sodiumphosphate, ethylene diamide tetraacetic acid (EDTA)) contains 3M sodiumchloride, 0.2M sodium phosphate, and 0.025 M EDTA. Other suitablemoderate stringency and high stringency hybridization buffers andconditions are well known to those of skill in the art and aredescribed, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, N.Y.(1989); and Ausubel et al., Short Protocols in Molecular Biology, 4thed., John Wiley & Sons (1999).

Alternatively, substantial complementarity exists when an RNA or DNAstrand will hybridize under selective hybridization conditions to itscomplement. Typically, selective hybridization will occur when there isat least about 65% complementary over a stretch of at least 14 to 25nucleotides, preferably at least about 75%, more preferably at leastabout 90% complementary. See M. Kanehisa, Nucleic Acids Res. 12:203(1984).

A “primer” used herein can be an oligonucleotide, either natural orsynthetic, that is capable, upon forming a duplex with a polynucleotidetemplate, of acting as a point of initiation of nucleic acid synthesisand being extended from its 3′ end along the template so that anextended duplex is formed. The sequence of nucleotides added during theextension process is determined by the sequence of the templatepolynucleotide. Primers usually are extended by a DNA polymerase.

“Ligation” may refer to the formation of a covalent bond or linkagebetween the termini of two or more nucleic acids, e.g., oligonucleotidesand/or polynucleotides, in a template-driven reaction. The nature of thebond or linkage may vary widely and the ligation may be carried outenzymatically or chemically. As used herein, ligations are usuallycarried out enzymatically to form a phosphodiester linkage between a 5′carbon terminal nucleotide of one oligonucleotide with a 3′ carbon ofanother nucleotide.

“Sequencing,” “sequence determination” and the like means determinationof information relating to the nucleotide base sequence of a nucleicacid. Such information may include the identification or determinationof partial as well as full sequence information of the nucleic acid.Sequence information may be determined with varying degrees ofstatistical reliability or confidence. In one aspect, the term includesthe determination of the identity and ordering of a plurality ofcontiguous nucleotides in a nucleic acid. “High throughput digitalsequencing” or “next generation sequencing” means sequence determinationusing methods that determine many (typically thousands to billions) ofnucleic acid sequences in an intrinsically parallel manner, e.g. whereDNA templates are prepared for sequencing not one at a time, but in abulk process, and where many sequences are read out preferably inparallel, or alternatively using an ultra-high throughput serial processthat itself may be parallelized. Such methods include but are notlimited to pyrosequencing (for example, as commercialized by 454 LifeSciences, Inc., Branford, Conn.); sequencing by ligation (for example,as commercialized in the SOLiD™ technology, Life Technologies, Inc.,Carlsbad, Calif.); sequencing by synthesis using modified nucleotides(such as commercialized in TruSeq™ and HiSeq™ technology by Illumina,Inc., San Diego, Calif; HeliScope™ by Helicos Biosciences Corporation,Cambridge, Ma.; and PacBio RS by Pacific Biosciences of California,Inc., Menlo Park, Calif), sequencing by ion detection technologies (suchas Ion Torrent™ technology, Life Technologies, Carlsbad, Calif);sequencing of DNA nanoballs (Complete Genomics, Inc., Mountain View,Calif.); nanopore-based sequencing technologies (for example, asdeveloped by Oxford Nanopore Technologies, LTD, Oxford, UK), and likehighly parallelized sequencing methods.

“Multiplexing” or “multiplex assay” herein may refer to an assay orother analytical method in which the presence and/or amount of multipletargets, e.g., multiple nucleic acid target sequences, can be assayedsimultaneously by using more than one probe, each of which has at leastone different detection characteristic, e.g., fluorescencecharacteristic (for example excitation wavelength, emission wavelength,emission intensity, FWHM (full width at half maximum peak height), orfluorescence lifetime) or a unique nucleic acid or protein sequencecharacteristic.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein comprises (anddescribes) embodiments that are directed to that value or parameter perse.

As used herein, the singular forms “a,” “an,” and “the” comprise pluralreferents unless the context clearly dictates otherwise. For example,“a” or “an” means “at least one” or “one or more.”

Throughout this disclosure, various aspects of the claimed subjectmatter are presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theclaimed subject matter. Accordingly, the description of a range shouldbe considered to have specifically disclosed all the possible sub-rangesas well as individual numerical values within that range. For example,where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the claimed subject matter. The upper and lowerlimits of these smaller ranges may independently be comprised in thesmaller ranges, and are also encompassed within the claimed subjectmatter, subject to any specifically excluded limit in the stated range.Where the stated range comprises one or both of the limits, rangesexcluding either or both of those comprised limits are also comprised inthe claimed subject matter. This applies regardless of the breadth ofthe range.

Use of ordinal terms such as “first”, “second”, “third”, etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements. Similarly, use of a), b), etc.,or i), ii), etc. does not by itself connote any priority, precedence, ororder of steps in the claims. Similarly, the use of these terms in thespecification does not by itself connote any required priority,precedence, or order.

VIII. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the present disclosure.

Example 1: FFPE Sample Preparation, De-Paraffinization, andDe-Crosslinking

A formalin-fixed, paraffin-embedded (FFPE) tissue sample was cut intothin tissue sections using a pre-warmed microtome. The sections wereplaced on slides and dried at a temperature higher than roomtemperature, followed by overnight drying at room temperature in adesiccator. Images of the sections were taken for record, e.g., in orderto monitor tissue morphology and/or detachment during sample processing.In some instances, images of the sections taken after de-crosslinkingwere compared to those before de-crosslinking.

The slides were baked uncovered in an oven, and then calibrated to roomtemperature. The sections were de-paraffinized and dehydrated usingxylene and absolute ethanol. The sections were then re-hydrated using anethanol series (e.g., 96% ethanol followed by 70% ethanol), and thenre-hydrated in nuclease free water (e.g., DEPC water). The slides weregently dried to allow application of a cassette to seal around thetissue sections, without letting the tissue section to dry completely.

The sections were then de-crosslinked using a catalyst disclosed hereinin a buffer solution. In some instances, the a de-crosslinking buffercan function as an antigen retriever buffer. Then the sections werewashed three times with RNase-free phosphate-buffered saline (PBS) orPBST (phosphate-buffered saline solution with a low-concentrationdetergent solution, e.g., 0.05% to 0.1% Tween™ 20) prior to applicationof labelling agents (e.g., detectable nucleic acid probes for nucleicacids and/or labelled antibodies for proteins) to the tissue samples.

Example 2: Analyte Detection in De-Crosslinked FFPE Human Breast CancerSamples

FFPE human breast cancer tissues were sectioned to 5 m thickness,transferred to Superfrost© Plus slides, and processed essentially asdescribed in Example 1. Specifically, 2-amino-5-methylbenzoic acid(Compound 1) was used as the de-crosslinking agent and applied tode-paraffinized and re-hydrated samples in a buffer solution, such as asolution of citrate buffer at pH 6 or a solution of Tris-EDTA (TE)buffer at pH 9. The samples were incubated under various de-crosslinkingtemperatures (e.g., 80° C. or 95° C.) for various time periods (e.g., 15minutes or 30 minutes). Different concentrations of the de-crosslinkingagent in the buffer solutions were tested, including 0 mM, 50 mM, 100mM, 200 mM, and 400 mM of Compound 1.

The de-crosslinked and washed tissue samples can be immediately used foranalyte detection. In some instances, circularizable probes (e.g.,padlock probes) targeting MALAT-1/ACTB (nuclear/cytoplasmic) orGAPDH/RPLPO (cytoplasmic) RNA transcripts were added in hybridizationbuffers (e.g., including SSC and formamide) and incubated with thesamples to allow hybridization of the circularizable probes to theirtarget nucleic acids. In addition to a target hybridization region, eachcircularizable probe also contained a common anchor region and a barcoderegion. Then, the probe hybridization mixture was removed and thesamples were washed. For ligation of the circularizable probeshybridized to their target nucleic acids, a ligation reaction mix (e.g.,containing a SplintR® ligase buffer, RNase inhibitor and SplintR®ligase) and a rolling circle amplification (RCA) primer were added tothe samples and incubated for probe circularization and RCA primerhybridization to the probes. The samples were washed and an RCA reactionmixture (containing Phi29 reaction buffer, dNTPs, Phi29 polymerase) wasadded and incubated for RCA of the circularized probes.

The samples were washed (e.g., in PBST) and detectable probes in ahybridization buffer (e.g., containing SSC and formamide) werehybridized to RCA products (RCPs) in the sample. The detectable probesincluded probes that hybridize to sequences (e.g., barcode sequences oranchor sequences) in the RCPs and comprise overhangs for hybridizationof fluorescently labelled detection oligonucleotides. The samples wereimaged in fluorescent microscope with 40× objective and the signalsassociated with the RCPs were quantified using a software. In someinstances, the samples were also stained with DAPI and/or labelledantibodies, e.g., fluorescently labelled anti-Vimentin antibody oranti-panCK antibody. In some instances, once the samples were imaged todetect signals associated with the detectable probes or labelledantibodies, the samples were stripped (e.g., using a denaturing agent)and contacted with additional detectable probes or labelled antibodiesfor the next imaging round.

FIG. 4 shows the detected object count per nucleic area (RCP count/μm²nuclei area) in cells of the human breast cancer samples treated withCompound 1 for de-crosslinking under various combinations of thede-crosslinking temperature (80° C. or 95° C.), time (15 minutes or 30minutes), buffer (Citrate or TE), and catalyst concentration (0 mM, 50mM, 100 mM, 200 mM, or 400 mM). The RCPs were detected using detectableprobes targeting the common anchor region in the RCPs. For instance,de-crosslinking for 30 minutes under 80° C., at 100 mM Compound 1 ineither the citrate buffer (pH 6) or the TE buffer (pH 9) appeared tosignificantly improve the detection of RCPs, as reflected by theincreased detected object counts per nucleic area as compared to control(de-crosslinked in a steamer for 30 minutes using citrate buffer (pH6)). In some instances, catalytic de-crosslinking appeared to boostsensitivity and RCP brightness using citrate buffer and between about100 and about 200 mM of Compound 1. RCP detection using detectableprobes targeting MALAT-1/ACTB and RCP detection using detectable probestargeting GAPDH/RPLPO showed similar results (data not shown).

FIGS. 5A-5B show representative images of anti-panCK antibody stainingin a control sample (de-crosslinked in a steamer) and in samplesde-crosslinked using the indicated de-crosslinking temperature, time,and catalyst concentration in either the citrate buffer (FIG. 5A) or theTE buffer (FIG. 5B). These results indicate that antibody staining(e.g., antibody positive signal intensity) may be improved by catalyticde-crosslinking with various concentrations of Compound 1 in citrate orTE buffer.

FIG. 6 shows representative DPAI images of the tissue samplesde-crosslinked in a thermal cycler (using citrate buffer, pH 6) atvarious combinations of temperature and time, either in the absence ofthe catalyst (0 mM) or using 200 mM of the catalyst. These resultsdemonstrate that at certain concentrations the de-crosslinking agent canimprove tissue integrity and/or adhesion (e.g., reduce waviness and/ortissue detachment from the substrate).

Together these results demonstrate that catalytic de-crosslinking (e.g.,at relatively lower temperatures such as 80° C.) can improve signaldetection of nucleic acids and proteins in FFPE human breast cancertissues in situ without compromising tissue integrity and/or adhesion.

Example 3: Analyte Detection in De-Crosslinked FFPE Samples from VariousTissues

FFPE human breast cancer, melanoma, lymph node, lung cancer, and normallung tissues were processed essentially as described in Example 1.Specifically, 2-amino-5-methylbenzoic acid (Compound 1) or(4-aminopyridin-3-yl)phosphonic acid (Compound 8) was used as thede-crosslinking agent and applied to de-paraffinized and re-hydratedsamples in a buffer solution, such as a solution of citrate buffer at pH6 or a solution of TE buffer at pH 9. The samples were incubated undervarious de-crosslinking temperatures (e.g., 80° C. or 95° C.) for 30minutes, using 0 mM or 200 mM of Compound 1 or Compound 8. Analytes inthe de-crosslinked tissue samples were detected essentially as describedin Example 2.

FIG. 7A shows representative images of the tissue samples analyzed usingdetectable probes targeting the common anchor region to detect the RCPs,demonstrating that catalytic de-crosslinking using either Compound 1 orCompound 8 enhanced visualization of transcripts GAPDH/RPLPO orMALAT-I/ACTB (data not shown) across the tissue types, as compared tode-crosslinking in a steamer.

FIG. 7B shows representative images of the tissue samples stained withan anti-Vimentin antibody. Catalytic de-crosslinking using eitherCompound 1 or Compound 8 enhanced anti-Vimentin antibody staining acrossthe tissue types, as compared to de-crosslinking in a steamer andde-crosslinking with heating at 95° C. in TE buffer.

FIG. 7C shows overlaid images of RNA detection and protein detection,demonstrating that catalytic de-crosslinking using either Compound 1 orCompound 8 enhanced RNA/protein visualization across the tissue types.

FIG. 8 shows the detected object count per nucleic area (RCP count/μm²nuclei area) in normal lung tissue samples. The RCPs were detected usingdetectable probes targeting barcode sequences for MALAT-I/ACTB orGAPDH/RPLPO or targeting the common anchor region in the RCPs(representative images in FIG. 8 , left panel). Higher RCP densities(detected object count per nucleic area (RCP count/μm² nuclei area))were observed in samples catalytically de-crosslinked using eitherCompound 1 or Compound 8, as compared to control samples de-crosslinkedin a steamer (data not shown). Catalytic de-crosslinking also improveddetected objected signal intensity above local background, as measuredby detected object signal intensity above local background (mean) inFIG. 8 , right panel.

Together these results demonstrate that catalytic de-crosslinking canimprove signal detection (e.g., signal counts as well as signal-to-noiseratios) of nucleic acids and proteins in situ across different tissuetypes including FFPE human breast cancer, melanoma, lymph node, lungcancer, and normal lung tissues.

Example 4: Analyte Detection in De-Crosslinked FFPE Mouse Brain Tissues

FFPE mouse brain tissues were processed essentially as described inExample 1, and gene expression analysis for RNA transcripts and proteinanalysis (e.g., anti-GFAP antibody staining) in the dentate gyrus (DG)region were performed essentially as described in Examples 2 and 3. Fourde-crosslinking agent and buffer combinations were tested: Compound 1 incitrate buffer (pH 6.8), Compound 15 in citrate buffer (pH 6.8),Compound 18 (trans-4-hydroxy-L-proline) in citrate buffer (pH 6.8), andCompound 1 in PBS (pH 7.4).

FIG. 9 shows the detected object signal intensity above local background(mean) in the mouse brain tissues, detected using detectable probestargeting the common anchor region in the RCPs. De-crosslinking withCompound 15 in citrate buffer (pH 6.8), Compound 18 in citrate buffer(pH 6.8), and Compound 1 in PBS (pH 7.4) all showed improved signaldensity compared to de-crosslinking with Compound 1 in citrate buffer(pH 6.8). Antibody staining was comparable among the de-crosslinkingagent and buffer combinations (data not shown).

Example 5: Analyte Detection in De-Crosslinked FFPE Samples withAdditives

FFPE human breast cancer samples sectioned to 5 m thickness wereprocessed essentially as described in Example 2. Specifically,2-amino-5-methylbenzoic acid (Compound 1) was used as thede-crosslinking agent and applied to de-paraffinized and re-hydratedsamples in a buffer solution with various additives, such as 0.05% SDS,0.2% SDS, 0.5% SDS, 0.05 M urea, 0.5 M urea, 0.2 μg/ml proteinase K, 0.5μg/ml proteinase K, 1 μg/ml proteinase K, and combinations thereof. Thesamples were incubated at 80° C. for 30 minutes, using 150 mM ofCompound 1. Analytes in the de-crosslinked tissue samples were detectedessentially as described in Example 2 using circularizable probes (e.g.,padlock probes) targeting ACTB, GAPDH, MALAT-1, RPLPO, Eef2, Ppib,POLR2A RNA transcripts. Detectable probes were hybridized to sequences(e.g., barcode sequences or anchor sequences) in the RCPs and comprisedoverhangs for hybridization of fluorescently labelled detectionoligonucleotides. The samples were imaged in fluorescent microscope with40× objective and the signals associated with the RCPs were quantifiedusing a software.

FIG. 10A-10C show the detected object count per nucleic area (RCPcount/μm² nuclei area) in cells of the human breast cancer samplestreated with Compound 1 for de-crosslinking in the presence or absenceof additives SDS, urea, and/or proteinase K as indicated.De-crosslinking with Compound 1 in the presence of SDS and proteinase K,or Compound 1 in the presence of urea and proteinase K appeared toimprove the detection of RCPs (e.g., decreased autofluorescence,increased puncta brightness and/or increased puncta numbers), asreflected by the increased detected object counts per nucleic area ascompared to control samples treated with Compound 1 with no additionaladditives or with single additives. In some instances, de-crosslinkingusing Compound 1 in the presence of both 0.5 M urea and 1 μg/mlproteinase K or Compound 1 in the presence of both 0.5 M urea with 0.5μg/ml Proteinase K showed increases in puncta brightness and/or numbersdetected. In addition, it was observed antibody staining (e.g., antibodypositive signal intensity for Pan cytokeratin, Vimentin, and Ki67) wascompatible with the catalytic de-crosslinking conditions with Compound 1in the presence of urea and proteinase K. Tissue morphology was alsoobserved to be preserved post workflow and detection as confirmed by H&Estaining that was performed.

The present disclosure is not intended to be limited in scope to theparticular disclosed embodiments, which are provided, for example, toillustrate various aspects of the present disclosure. Variousmodifications to the compositions and methods described will becomeapparent from the description and teachings herein. Such variations maybe practiced without departing from the true scope and spirit of thedisclosure and are intended to fall within the scope of the presentdisclosure.

1. A method for sample analysis, comprising: a) providing a biologicalsample immobilized on a substrate, wherein the biological sample isfixed; b) contacting the biological sample with a catalyst thatcatalyzes de-crosslinking of molecular crosslinks in the biologicalsample, wherein the catalyst is a compound of formula(I),

or a salt zwitterion, or solvate thereof, wherein: A is selected fromthe group consisting of —COOH, —P(═O)(OH)₂, and S(═O)₂OH; X¹, X², X³,and X⁴ are each independently selected from the group consisting of: CH,CR^(a), and N; each occurrence of R^(a) is independently selected fromthe group consisting of C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, —NO₂,—NR′R″, and —C(═O)NR′R″; and each occurrence of R′ and R″ isindependently selected from the group consisting of H and C₁₋₆ alkylwhich is optionally substituted with

wherein n1 is an integer from 12 to 16; c) contacting the biologicalsample with a labelling agent that directly or indirectly binds to aprotein analyte at a location in the biological sample, wherein thelabelling agent is conjugated to a reporter oligonucleotide comprising areporter sequence that identifies the labelling agent; and d) detectingan optical signal associated with the labelling agent or a productthereof, thereby detecting the protein analyte at the location in thebiological sample.
 2. The method of claim 1, wherein the molecularcrosslinks are products of one or more crosslinking agents.
 3. Themethod of claim 2, wherein the one or more crosslinking agents comprisean aldehyde, optionally wherein the crosslinking agent comprisesformaldehyde. 4-11. (canceled)
 12. The method of claim 1, wherein thecatalyst comprises one or more compounds selected from the groupconsisting of

or a salt, zwitterion, or solvate thereof.
 13. The method of claim 1,wherein the catalyst comprises

or a salt, zwitterion, or solvate thereof.
 14. The method of claim 1,wherein the catalyst comprises

or a salt, zwitterion, or solvate thereof.
 15. The method of claim 1,wherein the catalyst comprises

or a salt, zwitterion, or solvate thereof.
 16. The method of claim 1,wherein the catalyst comprises

or a combination thereof, or a salt, zwitterion, or solvate thereof.17-23. (canceled)
 24. The method of claim 1, wherein the biologicalsample is a tissue section. 25-58. (canceled)
 59. The method of claim 1,wherein a solution or a suspension comprising the catalyst and a bufferis contacted with the biological sample and the solution or suspensioncomprises sodium dodecyl sulfate (SDS), urea, and/or a proteinase.60-70. (canceled)
 71. The method of claim 1, wherein the labelling agentcomprises a binding moiety, wherein the binding moiety comprises anantibody or epitope binding fragment thereof.
 72. The method of claim 1,wherein the labelling agent comprises a detectable label.
 73. The methodof claim 1, wherein the reporter oligonucleotide comprises a barcodesequence. 74-87. (canceled)
 88. The method of claim 4, wherein theprotein analyte is an intracellular protein, a membrane-bound protein,or an extracellular protein. 89-90. (canceled)
 91. The method of claim1, wherein the optical signal is detected in situ in the biologicalsample.
 92. The method of claim 1, wherein the optical signal isdetected by imaging the biological sample.
 93. The method of claim 92,wherein the imaging comprises fluorescent microscopy. 94-119. (canceled)120. The method of claim 1, wherein the biological sample is contactedwith a probe that hybridizes to the reporter oligonucleotide of thelabelling agent to identify the protein analyte associated with thelabelling agent.
 121. The method of claim 1, wherein the labelling agentbinds indirectly to an intermediate probe comprising one or more barcodesequences.
 122. The method of claim 121, wherein the biological sampleis contacted with a detectably labeled tertiary probe that hybridizes tothe one or more barcode sequences of the intermediate probe.